Anchimeric radiometal chelating compounds

ABSTRACT

Anchimeric chelates are disclosed which are capable of rapidly forming stable chelates with radionuclide metals at or below physiological temperature. Bifunctional anchimeric chelates having these same properties are also disclosed which are useful for radiolabeling target specific molecules such as monoclonal antibodies and fragments thereof.

This application is a continuation application based on priorapplication Ser. No. 08/221,987, filed on Apr. 1, 1994, now abandoned,which is a continuation application based on prior application Ser. No.07/968,048, filed on Oct. 28, 1992, now abandoned, which was adivisional of prior application Ser. No. 07/157,284, filed Feb. 17,1988, now U.S. Pat. No. 5,202,451, issued Apr. 13, 1993.

TECHNICAL FIELD

This invention relates generally to chelating compounds which formcomplexes with metals such as radionuclide metals. Proteins such asantibodies may be radiolabeled by attaching the metal chelate compoundthereto. The chelating compounds are useful for producingradiodiagnostic and radiotherapeutic agents.

BACKGROUND OF THE INVENTION

Radiolabeled chelating compounds are useful both as medical diagnosticand therapeutic agents. For example, radiolabeled ethylenediaminetetraacetic acid (EDTA), and diethylenetriaminepentaacetate (DTPA) havebeen reported to be useful in evaluating renal functions, Klingensmithet al., J. Nucl. Med. 23:377 (1982). Similarly, Kasina et al., J. Med.Chem. 29:1933 (1986) report promising renal pharmaceuticals that aretechnetium chelates of N₂ S₂ diamido dimercaptides. Many otherradiolabeled diagnostic chelates have been reported and include:tartrate and orthophosphate, Molinski et al., U.S. Pat. No. 3,987,1571propylene amine oxime, Troutner et al., U.S. Pat. No. 4,615,876;polyhydroxycarboxylic acids, Adler et al., U.S. Pat. No. 4,027,005;organotrisubstituted trivalent phosphorus compounds, Dean et al., U.S.Pat. No. 4,582,700; bis-thiosemicarbazone, Vedee et al., U.S. Pat. No.4,564,472; gentisyl alcohol in combination with phosphonates, Fawzi,U.S. Pat. No. 4,232,000; mercaptoacetylglycylglycylglycine (MAG₃)Fritzberg et al., J. Nucl. Med. 27:111-116 (1986); mercaptocarboxylicacids, Winchell et al., U.S. Pat. No. 4,233,285; thiosaccharides,Kubiatowicz et al., U.S. Pat. No. 4,208,398; homocysteine andhomocysteinamide derivatives, Byrne et al., U.S. Pat. No. 4,571,430;metallothionein, Tolman, European application Apr. 10, 1984 0 137 457AZ; isonitrile, Jones et al., U.S. Pat. No. 4,452,774; andimidodiphosphonate, Subramanian et al., U.S. Pat. No. 3,974,268.

One class of such compounds is the bifunctional chelating compounds,which have a functional group capable of binding a metal and afunctional group reactive with a carrier molecule. Compounds of thistype are being actively investigated since they are capable of stablylinking radionuclides to target-specific biological molecules such asproteins, antibodies, and antibody fragments.

Diagnostic imaging of specific target tissue in vivo with aradiometal-chelate-antibody conjugate was reported by Khaw et al.,Science 209:295 (1980). Similarly, the therapeutic use ofradiometal-chelate-antibody conjugates to treat cellular disorders isdisclosed by Gansow et al., U.S. Pat. No. 4,454,106.

The procedure employed to insert a radiometal into a chelating compounddepends on the chemistry of the radiometal and the chemical structure ofthe chelating compound. A variety of radiometals can be incorporatedinto both simple and bifunctional chelating compounds. The particularradiometal selected depends on the intended application andavailability, as well as other factors.

Generally, radiometals intended for use as therapeutic agents are alpha,beta, or Auger electron emitters, such ¹⁰⁹ Pd, ¹¹¹ Ag, ¹¹⁹ Sb, ¹⁹⁸ Au,¹⁹⁹ Au, ⁶⁷ Cu, ¹⁰⁵ Rh, ¹⁸⁶ Re, ¹⁸⁸ Re, and ²¹² Bi. Radiometals intendedfor use as diagnostic agents are usually positron or gamma photonemitters. For example, in positron emission tomography ⁴³ Sc, ⁴⁴ Sc, ⁵²Fe, ⁵⁵ Co, and ⁶⁸ Ga can be employed, while for gamma camera imaging ²⁰³Pb, ⁹⁷ Ru, ¹⁹⁷ Hg, ⁶⁷ Ga, ²⁰¹ Tl, ^(99m) Tc, ^(113m) In, and ¹¹¹ In areusually selected.

Many of the radiometals described above are available in oxidationstates unsuitable for chelation without prior treatment. ^(99m) Tc, forexample, is available as pertechnetate (TcO₄) and must be reduced to alower oxidation state before chelation can occur. This is usuallyaccomplished by the addition of a reducing agent, such as Sn⁺² ordithionite at alkaline pH to the pertechnetate chelator mixture.

Transfer of the radiometal to the ultimate chelator is often facilitatedby employing a labile or weak chelating agent (WCA) in the reactionmixture, Fritzberg et al. (1986). In the case of ^(99m) Tc, for example,an initial complex may be formed with a WCA such as gluconate. The^(99m) Tc-gluconate complex forms quickly, thereby minimizingreoxidation of the ^(99m) Tc. Heating the initial Tc-WCA complex in thepresence of a strong chelating agent (SCA) results in transfer of ^(99m)Tc to the strong chelating agent in improved yields, compared tocarrying out the reduction of pertechnetate in the presence of thestrong chelator alone.

Pollack et al. British J. Hematology, 34:231 (1976) describe the kineticnature of the problem of transferring metals between strong chelatingagents. These authors demonstrate a significantly enhanced transfer ratewhen a weak chelating agent, such as nitrilotriacetate, is employed.

The need to enhance the transfer kinetics of a metal to a strongchelator is particularly important when the chelator is attached to aprotein. For example, Childs et al., J. Nucl. Med. 26:293-299 (1985)describe the rather harsh conditions, i.e. pH 4, necessary to achieveadequate binding of a radiometal to the antibody-bound chelator.Exposure to high temperatures or extremes of pH may denature orotherwise damage the protein to which the chelating compound isattached. Examples of weak chelating agents that have been used tofacilitate transfer of metals to proteins or strong chelating agentsattached thereto include the polyhydroxycarboxylates, glucoheptonate,Burchiel et al., J. Nucl. Med. 27:896 (1986) and tartrate, Kasina etal., Proc. Intl. Radio. Chem. Symp. 269-71 (1986). Strong chelatingagents that have been conjugated to target specific proteins include:DTPA, Childs et al. (1985); EDTA, Wieder et al., U.S. Pat. No. 4,352,751(1982); metallothionein, Tolman, European Patent Application 0137457(1985); bis-thiosemicarbozones, Arano et al., Int. J. Nucl. Med. Bio.12:425 (1986), U.S. Pat. No. 4,287,362; and diamido dimercaptide (N₂ S₂)Fritzberg et. al. (1986).

Even when a weak chelating agent is used to facilitate incorporation ofa radiometal into a strong chelating compound, the kinetics are notalways sufficient unless somewhat harsh conditions are employed. It isknown, for example, that transfer of technetium from a Tc-tartratecomplex to an antibody-N₂ S₂ conjugate is slow and requires heating to50° C. or more for an hour to effect acceptable radiometal transfer. SeeEuropean Patent Application Publication No. 188,256. Heating totemperatures above 37° C. often leads to aggregation of proteins such asantibodies, as well as nonspecific labeling of the antibody itself.

Accordingly, a need exists for a chelating compound that can rapidlyform stable chelates with radiometals at physiological temperatures orbelow. Radiolabeling of bifunctional chelators suitable for conjugationto target-specific biological molecules should be possible underconditions that preserve biological activity.

SUMMARY OF THE INVENTION

The present invention provides a chelating compound having a first siteat which a complex of a radionuclide metal forms and a second site atwhich a chelate of the radionuclide metal forms, wherein the complex hasa faster rate of formation and a lower thermodynamic stability than thechelate so that when the radionuclide metal is combined with thecompound, the complex at the first site forms initially, and theradionuclide metal subsequently is transferred to the second site toform a stable chelate. The radionuclide metal may be transferred to thesecond site by heating the compound to a temperature of about 37° C. orbelow after the initial complex has formed.

In one embodiment of the invention, the chelating compound has at itsfirst site two or more atoms chosen from oxygen, nitrogen, andphosphorous (in the form of oxides), arranged such that the atomsinteract with the radionuclide metal to form a complex. In oneembodiment of the invention, the chelating compound has as its secondsite a heteroatom chain containing at least four donor atoms chosen fromsulfur, nitrogen, and oxygen, wherein coordinate covalent bonds formbetween each of the donor atoms and the radionuclide metal to form achelate.

In a preferred embodiment of the invention, donor atoms of theheteroatom chain at the second site include at least one divalent sulfuratom and at least two nitrogen atoms, the sulfur atom being positionedat one terminus of the heteroatom chain, and from six to seven carbonatoms positioned so that at least two carbon atoms are located betweenany two of the donor atoms. One chelating compound of this invention hastwo nitrogen atoms and two sulfur atoms as the donor atoms.Alternatively, the chelating compound may have three nitrogen atoms andone sulfur atom as the donor atoms at the second site. It is preferredthat each sulfur donor atom has a protective group attached thereto. Inone embodiment of the invention, one or more of the sulfur atoms,together with a protective group attached thereto, defines a thioacetalor hemithioacetal group.

The chelating compound of this invention has a flexible divalent linker,linking the first and second sites. The linker generally comprises fromtwo to six methylene groups or methylene equivalents in a chain.

When the chelating compound is to be attached to a protein, thechelating compound additionally possesses a conjugation group that iscapable of reacting with a protein to bind the chelating compound to theprotein. The conjugation group is attached through a spacer to anycarbon or nitrogen atom of the chelating compound.

The invention includes a kit for producing a radiolabeled proteincomprising a chelating compound defined above and a protein to beradiolabeled. Optionally, the kit may comprise the protein and thechelating compound as a conjugate. The 1protein of the kit may be anysuitable target site-specific protein, such as an antibody or monoclonalantibody, or a fragment thereof.

The invention also includes a method of radiolabeling a proteincomprising the steps of: reacting a bifunctional chelating compound ofthe present invention with a protein to form a chelatingcompound-protein conjugate, then reacting the conjugate with aradionuclide metal to form a complex of the radionuclide metal at thefirst site of the chelating compound, and incubating the resultingconjugate-radionuclide metal complex at a temperature of 37° C. or lessto promote the transfer of the radionuclide metal to the second site,thereby forming a protein-bound chelate of the radionuclide metal. Analternative method for radiolabeling a protein comprises the steps ofreacting a chelating compound of the present invention with aradionuclide metal to form a complex of the radionuclide metal at thefirst site, incubating the complex to promote transfer of theradionuclide metal to the second site, thereby forming a chelate, thenreacting the compound with the protein, thereby producing achelate-protein conjugate.

Conjugates comprising a protein having a chelating compound (or aradiolabeled chelate) of the invention bound thereto also are disclosed.The radionuclide metal is selected from, for example, ^(99m) Tc, ¹⁸⁸ Re,¹⁸⁶ Re, ⁶⁷ Cu, ⁶⁴ Cu, ²¹² Pb, ²¹² Bi, and ¹⁰⁹ Pd. The protein may be anantibody, monoclonal antibody, or fragment thereof, such as a monoclonalantibody specific for cancer cells.

The present invention also provides a method of preparing a chelate of aradionuclide metal comprising reacting the radionuclide metal with thechelating compound described above to form a complex at the first site,then incubating the complex at a temperature of 37° C. or less topromote the transfer of the radionuclide metal to the second site,thereby forming the chelate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b illustrate the pathway for the synthesis of abifunctional diamidodimercapto anchimeric chelate.

FIGS. 2a and 2b illustrate the pathway for the synthesis of abifunctional amido amino dimercapto anchimeric chelate.

FIGS. 3a and 3b illustrate the pathway for the synthesis of abifunctional diamino dimercapto anchimeric chelate.

FIGS. 4a and 4b illustrate the pathway for the synthesis of abifunctional diamidodimercapto anchimeric chelate having an aromaticbridge.

DETAILED DESCRIPTION OF THE INVENTION

The novel compositions of the present invention are radiometal chelatesuseful for both diagnostic and therapeutic treatment. The chelatingcompounds have a first site capable of rapidly forming a labile complexwith a radionuclide metal and a second site capable of forming a stablecoordinate covalent chelate with a radionuclide metal. The radionuclidemetal complex that forms at the first site has a faster rate offormation but a lower thermodynamic stability than the radionuclidemetal chelate that forms at the second site. When a radionuclide metalis combined with the compound, a complex rapidly forms at the first siteand the radiometal is thereafter transferred to the second site, therebyforming a thermodynamically stable radiometal-chelate complex. In somesituations the chelating compound having first formed a complex with aradionuclide metal must be heated in order to transfer the radionuclidemetal to the second site. Heating of the compound to about 37° C. orless generally is sufficient to transfer the radiometal from the labilefirst site to the thermodynamically stable second site. The temperaturerequired to promote transfer of the metal from the first site to thesecond site will depend on such factors as the exact chemical structureof the chelating compound, as discussed more fully below. One advantageof compounds of the instant invention is that they rapidly formthermodynamically stable complexes with radiometals without thenecessity of heating above physiological temperatures. It is believedthat these compounds exhibit this unusual property because of theneighboring group effect. Compounds exhibiting this effect haveneighboring functional groups that participate in the overall reactionand exhibit more rapid kinetics than compounds without reactiveneighboring functional groups.

The compounds of the instant invention can be viewed as molecules havingboth a weak complexing site (Site 1) and a strong chelating site (Site2) covalently linked together. It is believed that when this compound ismixed with a metal M, a relatively labile complex rapidly formsaccording to Equation (1) below. ##STR1## Subsequently, when sufficientheat is applied (or when incubated at room temperature in some cases),the weak complexing moiety of the molecule releases the metal to thestrong chelating moiety, resulting in rearrangement of the complexaccording to Equation (2) below. ##STR2## It is unlikely that the metalis actually released into solution during the rearrangement representedby Equation (2); rather the rearrangement is likely to occur withanchimeric assistance. (See W. Page, Chem. Soc. Rev., 2, 295-323 (1973);and J. March, Advanced Organic Chemistry, McGraw-Hill, New York, 2dedition, p. 280, for discussions of anchimeric assistance.)

The functional groups present at the first site (Site 1) forming theweak complex are generally groups comprising two or more atoms selectedfrom oxygen, nitrogen, or phosphorous (in the form of oxides such asphosphate or phosphonate). Preferred functional groups present at thefirst site comprise hydroxyl groups or amino groups, either alone or inconjunction with carboxyl groups. The portion of the compounds of theinvention which functions as the first site may be selected from, forexample, iminodiacetate, alkyl phosphonate, alkyl diphosphonate,N-glycine, aminoalkylpolyacetate, alkylhydroxycarboxylate,polyhydroxyaminoalkanes, alkylaminohydroxycarboxylate, andalkyldihydroxydicarboxylate. Preferred substituents Q includesubstituents selected from the group consisting ofpolyhydroxycarboxylates, gluconate, tartrate, alkyl phosphonate, alkyldiphosphonate, gluconamide, N-glycine, N-3-aminopropanoate, α-hydroxyacids, α-hydroxy-β-amino acids (e.g., α-hydroxy-β-amino propanoate),α-hydroxy-α-amino acids, deoxyamino uronic acids (e.g.,6-amino-6-deoxy-D-gluconic acid), β-diketones or their enol equivalents,##STR3## wherein R is C₁ -C₅ lower alkyl and R₁₇ is selected fromhydrogen, lower alkyl, or R₁₅ --Z, wherein R₁₅ represents a divalentspacer and Z represents a group reactive with a protein, as describedbelow under Formula I. Other compounds having suitable functional groupsto form the initial complex at the first site include derivatives of theabove groups.

The chelating compounds of the instant invention generally contain, atthe second site, a heteroatom chain containing at least four donor atomschosen from sulfur, nitrogen, and oxygen. These donor atoms are capableof forming coordinate covalent bonds with a radionuclide metal so that athermodynamically stable radionuclide metal-chelate forms at the secondsite. It is preferred that the donor atoms include at least one divalentsulfur atom and at least two nitrogen atoms and that the sulfur atom ispositioned at one terminus of the heteroatom chain. In addition to theheteroatoms, the heteroatom chain contains from 6 to 7 carbon atomspositioned such that at least two of the carbon atoms are interposedbetween any two of the donor atoms. In one embodiment of the invention,the donor atoms in the heteroatom chain are two nitrogen atoms and twosulfur atoms. Chelating compounds of this type are referred tohereinafter as N₂ S₂ compounds. The sulfur atoms in this compound aregenerally divalent sulfur and the nitrogen atoms are independentlyeither amide or amine nitrogen. In an alternative embodiment of theinstant invention, the donor atoms in the heteroatom chain of the secondsite consist of three nitrogen atoms and one sulfur atom. Compounds ofthis type are referred to hereafter as N₃ S compounds in which thesulfur atom is divalent sulfur and the nitrogen atoms are independentlyamide or amine nitrogen.

The second site of the chelating compound is the strong chelating moietyof the molecule. Exemplary compounds forming strong coordinate covalentchelates with radionuclide metals which may be used as the strongchelating moiety are N₂ S₂ compounds such as thebis(mercaptoalkanoamido)alkanoic acids. Examples of these are2,3-dimercaptoacetamidobutanoate, and 4,5-dimercaptoacetamidopentanoate,generally referred to as CO₂ -DADS compounds, as well as1,2-dithioacetamidoethane (DADS), 4,5-dithioacetamidopentanoic acid, andamino-amides such as 4-thioacetamido-5-thioethylaminopentanoic acid.These compounds can be synthesized by procedures described in FritzbergU.S. Pat. No. 4,444,690, herein incorporated by reference, EuropeanPatent Application Publication No. 188,256, and copending U.S. patentapplication Ser. No. 065,017, filed Jun. 19, 1987 now U.S. Pat. No.5,175,343. Exemplary N₃ S compounds that form strong coordinate covalentchelates with radionuclide metals are mercaptoacetylglycylglycylglycine(MAG₃) compounds synthesized by procedures described in Fritzberg etal., J. Nucl. Med. 27:111-116 (1986), herein incorporated by reference,and European Patent Application Publication No. 173,424. Suitable MAG₃compounds include not only MAG₃ per se, but also analogous compounds inwhich the terminal glycine is replaced by a β, γ, or δ amino acid, forexample mercaptoacetylglycylglycyl-γ-aminobutyrate.

It is preferred that each terminal sulfur atom in the heteroatom chainof the second site be conjugated with a protective group. Thesulfur-protective groups may be varied widely, being acyl groups, thiogroups or other compounds which provide protection of the thio groupduring subsequent manipulations. The sulfur-protective groups also serveto stabilize the chelating compounds by preventing reaction of thesulfurs with groups that are part of the chelating compound itself. Forexample, if the protecting groups are replaced with hydrogens, thesulfurs may displace an active ester protein conjugation group from thechelating compound.

Illustrative sulfur-protective groups include benzoyl, acetyl,acetamidomethyl, m- or p-phthaloyl, thioglycolic, o-carboxythiophenol,ethylthiocarbonate, β-mercaptopropionic, tetrahydropyranyl, ethoxyethyl,sulfonato, acetamidomethyl, etc. Alternatively, cyclic di- orpolysulfides may be formed. Disulfides may be prepared using sulfinylhalides, dinitrothiophenoxide-substituted mercaptans, with mildoxidation in the presence of excess of the protective group, etc.

The protective groups may be removed in a variety of ways. Thioestersmay be hydrolyzed using aqueous ammonia, sodium alkoxide in alkanol, orany conventional technique. Disulfides may be cleaved withdithiothreitol, glutathione, β-mercaptoethylamine, or other conventionalreagent. Cleavage of the disulfide may occur prior to or afterconjugation to the polypeptide.

In one embodiment of the invention, the sulfur-protecting group, whentaken together with the sulfur atom(s) to be protected, represents athioacetal or hemithioacetal. These sulfur-protective groups aredisplaced during the radiolabeling reaction, conducted at acidic pH, inwhat is believed to be metal-assisted acid cleavage; and covalent bondsform between the sulfur atoms and the radionuclide metal. Advantages ofthese sulfur-protective groups include the fact that a separate step forremoval of the sulfur-protective groups is not necessary. Theradiolabeling procedure thus is simplified, which is especiallyadvantageous when the chelating compounds are to be radiolabeled in ahospital laboratory shortly before use. In addition, the basic pHconditions and harsh conditions associated with certain knownradiolabeling procedures or procedures for removal of othersulfur-protective groups are avoided. Thus, base-sensitive groups on thechelating compound survive the radiolabeling step intact. Suchbase-labile groups include any group which may be destroyed, hydrolyzed,or otherwise adversely affected by exposure to basic pH. In general,such groups include esters, Michael-type acceptors (e.g., maleimides)and isothiocyanates, among others. The use of thioacetal orhemithioacetal protective groups, therefore, is especially advantageouswhen the chelating compound is radiolabeled prior to conjugation of theresulting chelate to a protein (the "pre-formed" approach, describedbelow), since base-labile protein conjugation groups survive theradiolabeling procedure. Another advantage is that these protectivegroups (especially the hemithioacetals) are relatively easily displacedfrom the compound, so that the chelating compounds of the invention maybe radiolabeled under physiologically acceptable conditions of pH andtemperature.

Thioacetals and hemithioacetals which may be used in the presentinvention include those groups which effectively maintain the sulfurs ina nonreactive state until the radiolabeling step, at which time theprotective groups are displaced in the presence of the metallicradioisotope under acidic conditions. In general, the hemithioacetalS-protecting groups are somewhat more acid labile in the radiolabelingreaction than the thioacetal groups, and therefore are generallypreferred.

When hemithioacetal protective groups are used, each sulfur atom to beprotected has a separate protective group attached to it, which,together with the sulfur atom, defines a hemithioacetal group. Preferredhemithioacetals generally are of the following formula, wherein thesulfur atom is a sulfur atom of the chelating compound, and a separateprotecting group is attached to each of the sulfur atoms on thechelating compound: ##STR4## wherein R³ is a lower alkyl group,preferably of from two to five carbon atoms, and R⁴ is a lower alkylgroup, preferably of from one to three carbon atoms. Alternatively, R³and R⁴ may be taken together with the carbon atom and the oxygen atomshown in the formula to define a nonaromatic ring, preferably comprisingfrom three to seven carbon atoms in addition to the carbon and oxygenatoms shown in the formula. R⁵ represents hydrogen or a lower alkylgroup, wherein the alkyl group preferably is of from one to three carbonatoms. Examples of such preferred hemithioacetals include, but are notlimited to: ##STR5##

When the chelating compound comprises two or more sulfur donor atoms,the sulfur-protective groups may be the same or different. In somecases, the compounds may be synthesized more easily when a differentprotective group is attached to each sulfur donor atom. For example, achelating compound having two sulfur donor atoms may contain onehemithioacetal sulfur-protecting group and one acetamidomethyl surfurprotecting group.

The chelating compound of the instant invention also contains a flexibledivalent linker which connects the labile complexing first site to thethermodynamically stable chelating second site. It is preferred that theflexible linker comprise from about two to about six methylene groups orequivalents comprising covalent σ-bonds and may contain one or more ofthe following: ether, thioether, amine or amide groups. Methylene chainsor equivalents of this length are of suitable length and flexibility toallow transfer of a radionuclide metal from the first site to the secondsite via first-order kinetics characteristic of compounds exhibiting thenearest neighbor effect.

The chelating compounds of the present invention can be synthesized bycovalently linking compounds which form strong coordinate covalentchelates with radionuclide metals to compounds known to rapidly formlabile complexes with radionuclide metals. Covalent linking of thecompounds through the flexible divalent linker can be achieved byconventional procedures which will vary according to the chemicalstructure of the compounds. For example,2,3-dimercaptoacetamidopropanoate, having the terminal sulfurs protectedwith either ethoxyethyl or acetamidomethyl, can be reacted withN-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide. The resultingactive ester can then be reacted with 6-amino-6-deoxy-D-gluconic acid toproduce2,3-di(S-ethoxyethylmercaptoacetamido)-propanoyl-6-amino-6-deoxy-D-gluconicacid. Other exemplary monofunctional (as opposed to bifunctional)anchimeric chelating compounds produced analogously include4,5-di(S-ethoxyethylmercaptoacetamido)pentanoyl-6-amino-6-deoxy-D-gluconicacid and3,4-di(S-ethoxyethylmercaptoacetamido)butanoyl-6-amino-6-deoxy-D-gluconicacid.

Compounds of this type are particularly useful for replacingradionuclide metal-chelate systems that form highly stable complexes butwith slow kinetics. These are exemplified by certain Tc and Re diamidodimercapto complexes in which disulfide formation, oxidation, etc., andcluster formation (i.e., one metal atom associated with more than onechelating compound) restricts the chelation kinetics to relatively slowrates. However, once formed, such complexes are particularly stable.Such complexes have found practical use as bifunctional chelates forlabeling proteins (antibodies). Due to the slow kinetics of chelation,commercial application has been limited to the pre-formed chelateapproach (described below) (see copending U.S. patent application Ser.No. 065,017, filed Jun. 19, 1987, now U.S. Pat. No. 5,175,343 entitled"Metal Radionuclide Labeled Proteins for Diagnosis and Therapy"), inwhich the slowness can be overcome by heating at the small moleculestage (e.g., at temperatures of about 75° C.) to form the radiolabeledchelate, and then conjugating the chelate to the protein at roomtemperature. This approach is much more complex than a post-formapproach (described below) in which the metal specifically forms a N₂ S₂type chelate in a chelating compound already conjugated to the antibody.Successful application that avoids nonspecific, uncontrolled binding ofTc and Re to the protein provides a much simpler labeling and, hence,product formulation. The use of the rapid complex formation at the firstsite to bring the metal into position to accelerate N₂ S₂ chelationprovides a means to achieve more rapid chelation of the metalradionuclide than association with nonspecific binding sites on theantibody.

Compounds of the instant type are particularly useful for chelatingradionuclide metals having diagnostic or therapeutic use. Theseradiometals include, but are not limited to, ^(99m) Tc, ¹⁸⁸ Re, ¹⁸⁶ Re,⁶⁷ Cu, ⁶⁴ Cu, ²¹² Pb, ²¹² Bi, ¹⁰⁵ Rd, ⁹⁷ Ru, and ¹⁰⁹ Pd. Methods forpreparing these isotopes are known. Molybdenum/technetium generators forproducing ^(99m) Tc are commercially available. Procedures for producing¹⁸⁶ Re include the procedures described by Deutsch et al., Nucl. Med.Biol., 13:4:465-477, (1986) and Vanderheyden et al., InorganicChemistry, 24:1666-1673, (1985), and methods for production of ¹⁸⁸ Rehave been described by Blachot et el., Intl. J. of Applied Radiation andIsotopes, 20:467-470, (1969) and by Klofutar et el., J. ofRadioanalytical Chem., 5:3-10, (1970). Production of ¹⁰⁹ Pd is describedin Fawwaz et el., J. Nucl. Med., 25:796 (1984). Production of ²¹² Pb and²¹² Bi is described in Gansow et al., Amer. Chem. Soc. Symp. Ser.,241:215-217 (1984), and Kozah et el., Proc. Nat'l. Aced. Sci. USA,83:474-478 (1986).

The method for preparing a radionuclide metal-chelate, according to thepresent invention, comprises reacting a radionuclide metal of the typedescribed above with the instant chelating compound to form a complex atthe first site, essentially as depicted in Equation (1) above. Theconditions suitable for carrying out this reaction are known to thoseskilled in the art and depend both on the type of radiometal to bechelated and its normal valence. For example, to prepare the technetiumchelate, the chelating compounds of the instant invention may becombined with a pertechnetate solution in the presence of a reducingagent (e.g., stannous ion or dithionite under conventional conditions),whereby the technetium complex is formed at the first site as a stablesalt. The corresponding rhenium complex may be formed by reducingperrhenate with stannous ion or dithionite. Site 1 complexes of ²¹² Pb,²¹² Bi and ¹⁰⁹ Pd may be prepared by simply combining the appropriatesalt of the radionuclide metal with the chelating compound. It is notnecessary to treat the lead, bismuth, palladium, and copper isotopeswith a reducing agent prior to complexation because such isotopes arealready in an oxidation state suitable for complexation (see forexample, Fritzberg et al., (1986)). After the initial complex hasformed, it is incubated at mild temperatures to promote transfer of theradionuclide metal to the second site, thereby forming athermodynamically stable chelate, essentially as depicted in Equation(2) above. The preferred compounds of the instant invention are thosethat require heating to 37° C. or less to effect the transfer of theradiometal from the first site to the second site.

The temperature required to promote the transfer of the radionuclidemetal from the first site to the second site will vary according to suchfactors as the exact chemical structure of the chelating compound. Forexample, certain sulfur-protecting groups are displaced at lowertemperatures during radiolabeling than are others. Also, slightly highertemperatures may be required to promote transfer of the radiometal whenthe chelating compound comprises one or more amide groups (i.e., acarboxyl group adjacent to a nitrogen donor atom, as shown below). Forsome chelating compounds of the present invention, incubation at roomtemperature is expected to be sufficient to promote transfer of theradionuclide metal to the second site, which binds the radionuclidemetal in the form of a strong, stable chelate. In general, theradiolabeling reaction mixture is not cooled, but is incubated at atemperature between ambient temperature and about 37° C. for a length oftime sufficient to promote transfer of the metal to the second site,thus forming the chelate.

Optionally, a chelating compound of the instant invention additionallycomprises a conjugation group that is capable of reacting with a proteinthereby binding the chelating compound to the protein. Chelatingcompounds of this type are hereinafter referred to as bifunctionalchelating compounds. The conjugation group is generally attached througha spacer group to any carbon or nitrogen atom of the chelating compound.A variety of functional groups suitable for conjugation to a protein inan aqueous reaction medium under conditions that preserve the biologicalactivity of the protein may be employed. Among the suitable conjugationgroups are those selected from the group consisting of esters, activeesters, halomethyl ketones, maleimide groups, other Michael-typeacceptors, free amines, and isothiocyanate groups. Proteins contain avariety of functional groups (e.g., carboxylic acid or free aminegroups) which are available for reaction with the protein conjugationgroup on the chelating compound. One or another of the groups may bepreferred, depending upon the particular radionuclide metal, theprotein, the chelating compound, and the conditions for conjugation. Asused herein, the term "aqueous medium" is meant to include not onlytotally aqueous media but also mixed aqueous/organic media, wherein theorganic component is present only in a relatively low concentration;i.e., a concentration low enough to minimize damage to polypeptides(e.g., denaturation).

A variety of esters may be used as the protein conjugation group,including aromatic esters containing electron-withdrawing groups, orα-substituted methyl esters (in which the substituents areelectron-withdrawing groups, such as, but not limited to, --CH₂ CN,--CH₂ --CO--CH₂ CH₃ or CH₂ --CO--CH₃).

Preferred esters for use in the present invention have severalstructural features that impart the desired stability and reactivity tothe esters. For example, preferred esters should be relatively stable,especially with respect to hydrolysis in aqueous solutions. Chelatingcompounds comprising such esters may be added to aqueous reactionmixtures or mixed aqueous/organic reaction mixtures (i.e., forradiolabeling or for protein conjugation reactions) with relativelylittle hydrolysis of the ester group. Thus, such hydrolysis-resistantesters are particularly useful in reactions with proteins, since suchreactions preferably are conducted under aqueous conditions to preventdenaturation of the proteins that may occur in organic solvents.Advantageously, the ester is sufficiently stable to allow preparation ofthe chelating compound ahead of time and storage, even under humidconditions, until needed, with the ester group remaining substantiallyintact.

The term "active ester" is known to refer to esters that are highlyreactive in nucleophilic substitution reactions. Preferred active estersfor use in the present invention are highly reactive toward groups onpolypeptides so that the active ester-containing chelate compounds arebound to the polypeptides through the reaction. Reaction of an esterwith a free amine group (present on lysine residues) on a proteinproduces an amide bond. These active esters comprise leaving groups(i.e., the --OR' portion of an ester R--CO--OR') that are sufficientlyelectron-withdrawing to increase the susceptibility of the carbonyl toattack by nucleophilic groups on the protein. The kinetics of thereaction preferably are such that the ester reacts quickly withnucleophilic groups on the polypeptide. Thus, the free unreacted estergroups, potentially susceptible to hydrolysis (especially if thereaction is conducted at a basic pH), are subjected to the aqueousreaction conditions for only a short time. Hydrolysis of the ester,therefore, is further minimized, and a relatively high ratio of thedesired reaction to hydrolysis of the ester results.

Common esters that find use are the o- and p-nitrophenyl,2-chloro-4-nitrophenyl, cyanomethyl, 2-mercaptopyridyl,hydroxybenztriazole, N-hydroxysuccinimide, trichlorophenyl,tetrafluorophenyl, 2-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl,o-nitro-p-sulfophenyl, N-hydroxyphthalimide, N,N-diethylamino,N-hydroxypyrrolidone, tetrafluorothiophenyl, and equivalents. For themost part, the esters will be activated phenols, particularlynitro-activated phenols and cyclic compounds based on hydroxylamine. Asother hydroxylic compounds become available, these also may find use inthis invention.

Especially good results are achieved by using a2,3,5,6-tetrafluorophenyl ester, which is an active ester having theabove-described properties of stability and high reactivity. Anotherester group exhibiting high reactivity toward proteins is a thiophenylester.

The use of esters comprising nitro groups may be disadvantageous incertain circumstances. For example, the nitro group may be reduced bystannous ion that may be present when the stannous ion is added as apertechnetate or perrhenate reducing agent, as described above.

Alternatively, the protein and/or chelating compound may be derivatizedto expose or attach additional reactive functional groups. Thederivatization may involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company,Rockford, Ill. (See the Pierce 1986-87 General Catalog, pages 313-354.)Alternatively, derivatization may involve chemical treatment of theprotein, e.g., glycol cleavage of the sugar moiety of a glycoproteinantibody with periodate to generate free aldehyde groups. The freealdehyde groups on the antibody may be reacted with free amine orhydrazine groups on the chelating compound to bind the agent thereto.(See U.S. Pat. No. 4,671,958.) Procedures for generation of freesulfhydryl groups on antibodies or antibody fragments also are known.The sulfhydryl groups are reactive with maleimide and amino groups. (SeeU.S. Pat. No. 4,659,839.)

The protein to which the chelating compound is to be attached may bevaried widely, depending upon the nature of the use of the radionuclidemetal. In general, the protein delivers the chelated radionuclide to adesired target site in vivo. Suitable proteins include, but are notlimited to, receptors, hormones, lymphokines, growth factors,substrates, and particularly compounds binding to surface membranereceptors, where the complex may remain bound to the surface or becomeendocytosed. Among receptors are surface membrane receptors, antibodies(including monoclonal antibodies) enzymes, naturally occurringreceptors, lectins, and the like. Of particular interest areimmunoglobulins or their equivalent, which may be whole antibodies orfragments thereof, e.g., Fab, Fab', F(ab')₂, or F_(v) fragments, orT-cell receptors, etc. As used herein, the term "protein" includespolypeptides, proteins, or fragments thereof. These proteins andpolypeptides may be modified, provided the biological activity necessaryfor the intended diagnostic or therapeutic application of theradiolabeled polypeptide is retained. For example, a modified antibodyor fragment thereof may be used as long as binding to the desiredantigen still occurs. The amino acid sequence of a protein may be varied(e.g., by known mutation techniques or deletion of portions thereof) aslong as the desired biological activity (e.g., binding of the protein tospecific target cells, tissues, or organs) is retained. Methods ofmodifying proteins also may include, among others, attachment ofbifunctional linker compounds that react with both a group on a proteinand with the Z group on the chelating compounds, thereby binding thechelating compound to the protein through the linking compound. Theprotein may be purified from a natural source or may be synthetic (e.g.,produced by recombinant DNA technology or chemical synthesisprocedures).

Proteins that bind to the desired target site are said to be "targetspecific." For example, antibodies that bind to a particular antigen aresaid to be target specific for that antigen. It is to be understood thatsuch proteins or antibodies are rarely 100% specific for a target site,and a certain degree of cross-reactivity with other tissues is common.An example of a target site is a cancer cell. Many antigens associatedwith various types of cancer cells have been identified, and monoclonalantibodies specific for a number of these cancer cell-associatedantigens also are known. Among the many such monoclonal antibodies whichmay be used are anti-TAC, or other interleukin 2 receptor antibodies;9.2.27 and NR-ML-05 to the 250 kilodalton human melanoma-associatedproteoglycan; NR-LU-10 to 37-40 kilodalton pancarcinoma glycoprotein;NR-CO-02 to carcinoembryonic antigen (CEA) and colon carcinoma; NR-CE-01to CEA; and OVB₃ to an as yet unidentified cancer-associated antigen.Antibodies derived through genetic engineering or protein engineeringmay be employed as well. Such antibodies are examples of the many targetspecific proteins suitable for use in accordance with the presentinvention.

An example of a chelating compound of the present invention isrepresented by structural Formula I. ##STR6## 1) Wherein: a) T is asulfur-protecting group;

b) R₀ is S--T, carboxylic oxygen or ##STR7## c) the R groups designatedR₁ through R₁₁ are independently selected from COOH, R₁₄ and R15--Z,wherein any two of said R groups, when bonded to the same carbon atom,may be taken together to form an oxo group, with the provisos that:

i) R₇ and R₈ taken together, and R₉ and R₁₀ taken together, are not bothsimultaneously oxo;

ii) R₂ and R₃ taken together, and R₄ and R₅ taken together, are not bothsimultaneously oxo;

iii) R₄, R₅, R₉, and R₁₀ may all be taken together to form a hydrocarbonring;

iv) R₆ is hydrogen when either R₂ and R₃ or R₄ and R₅ represent an oxogroup;

v) R₁₁ is hydrogen when either R₇ and R₈ or R₉ and R₁₀ represent an oxogroup;

vi) each of R₇, R₈, R₉, and R₁₀ are hydrogen when R₁₁ is R₁₅ --Z or R₁₆;

vii) each of R₂, R₃, R₄, and R₅ are hydrogen when R₆ is R₁₅ --Z or R₁₆ ;

viii) R₆ and R₁₁ cannot be COOH;

d) R₁₂ and R₁₃ are (1) independently R₁₄ or R₁₅ --Z when R₀ is eitherS--T or ##STR8## or (2) taken together to form oxo when R₀ is eithercarboxylic oxygen or ##STR9## with the proviso that when R₁₂ and R₁₃ areoxo and R₀ is ##STR10## and one of these two R₁ groups is R₁₅ --Z orR₁₆, the other R₁ group is hydrogen;

e) R₁₄ is hydrogen, lower alkyl, or R₁₆ ;

f) n is 0 or 1 provided that n equals 1 not more than once;

g) R₁₅ is a divalent spacer selected from substituted or unsubstitutedlower alkyl, which may additionally comprise one or more groups selectedfrom --O--, --NH--, --NR--, --CO--, --CO₂ --, --CONH--, --S--, --SO--,--SO₂ --, --CO₂ NH--, and --SO₂ NR--, where R is selected from C₁ -C₃alkyl;

h) Z is a functional group suitable for reacting with a protein, proteinfragment, or polypeptide under conditions that preserve biologicalactivity of the protein, protein fragment, or polypeptide;

2) wherein said compound comprises at least one substituent R₁₅ --Z; and

3) wherein at least one of the groups R₁ -R₁₄ is substituent R₁₆ havingthe formula: ##STR11## wherein: a) one or more R₁₇ groups are bonded toany carbon or nitrogen atom and are selected from the group hydrogen,lower alkyl, or R₁₅ --Z;

b) X is a radical selected from the group consisting of fullysubstituted carbon, nitrogen, oxygen, sp² or sp carbon, or amide orimide nitrogen;

c) p in an integer, 2, 3, 4, 5, or 6 provided that:

i) when p is 2 or 3, X is fully substituted carbon;

ii) when p is 4, X is fully substituted carbon, nitrogen, or oxygen,further provided that at least three radicals X are fully substitutedcarbon;

iii) when p is 5 or 6, at least three radicals X are fully substitutedcarbon and further provided that only one radical X may be sp² or spcarbon, amide or imide nitrogen; and

d) Q represents the first site.

In compounds of the type represented by Formula I, Q may be selectedfrom among the compounds and structures described above as beingsuitable for use as the first site, i.e., the weak complexing moiety ofthe chelating compound of the invention. The substituent R₁₅ --Z ispresent when the chelating compound is to be attached to a protein. TheZ group may be chosen from the protein conjugation groups describedabove.

The chelating compounds represented by Formula I comprise, at the secondsite, a heteroatom chain containing four donor atoms chosen from sulfur,oxygen, and nitrogen. Two of the donor atoms are nitrogen, independentlyeither amino or amido. One of the donor atoms positioned at one end ofthe heteroatom chain is sulfur, and the other donor atom, located at theother end of the heteroatom chain, is either carboxylic oxygen, sulfur,or amino nitrogen. Accordingly, the compounds represented by Formula Iare referred to by their donor atom composition at the second site;namely, N₂ SO, N₂ S₂, and N₃ S, respectively.

In addition to the donor atoms, there are 6 or 7 carbon atoms in theheteroatom chain that comprises the second site. The carbon atoms arepositioned so that at least two carbons interpose between any two donoratoms of the heteroatom chain. When two carbon atoms are positionedbetween two donor atoms, they, together with a radionuclide metal,define a 5-member ring in a portion of the heteroatom chain. Five-memberrings are preferred at the second site because this planer structureallows formation of the most stable chelate with a radionuclide metal.Accordingly, the most preferred chelate compound represented by Formulais produced when n equals zero in each case or the heteroatom chaincomprises a total of 6 carbon atoms. This embodiment produces aparticularly stable chelating compound in which the heteroatom chain,together with the radionuclide metal, defines three 5-membered rings.

Suitable chelating compounds are obtained when n equals 1 in Formula I,provided that n equals 1 only once or the heteroatom chain comprises atotal of 7 carbon atoms. In this embodiment, two donor atoms areseparated by three carbon atoms that, when taken together with theradionuclide metal, defines a 6-member ring in a portion of theheteroatom chain. Accordingly, when four donor atoms and seven carbonatoms comprise the heteroatom chain of the second site, these atoms,together with the radionuclide metal, define two 5-membered rings andone 6-membered ring. Chelating compounds of the type represented byFormula I, containing more than one 6-member ring at the second site,are generally not suitably stable unless donor atoms at the first sitealso participate in chelation of the radionuclide metal.

The chelating compounds represented by Formula I have a flexibledivalent linking group that links the first site with the second site.This linking group, defined as (X)_(p) in Formula I, may be from about 2to 6 methylene equivalents in length and must possess sufficientflexibility to allow intramolecular transfer of a radionuclide metalfrom the first site to the second site by heating to 37° C. or less.Accordingly, when p in Formula I is 2 or 3, radical X must be fullysubstituted carbon, nonlimiting examples of which are: --CH₂ --CH₂ --and --CH₂ --CH₂ --CH₂ --. The hydrogens of these exemplary compounds maybe independently substituted with other groups, provided the carbonatoms remain fully substituted.

When p is 4, X must be fully substituted carbon, nitrogen, or oxygen,provided at least three radicals X are fully substituted carbon.Nonlimiting examples of these are: --CH₂ --CH₂ --CH₂ --CH₂ --, --CH₂--O--CH₂ --CH₂ --, and --CH₂ --CH₂ --NH--CH₂ --. Fully substitutedoxygen and nitrogen may be located in any position in these exemplarycompounds and, as above, the hydrogens may be independently substitutedwith other groups, provided that radical X remains fully substituted.

When p is 5 or 6, at least 3 radicals X must be fully substitutedcarbon. The other radicals may be fully substituted carbon, nitrogen, oroxygen, and one of the other radicals may be sp² or sp carbon, amide orimide nitrogen. Nonlimiting examples of these are: --CH₂ --CH₂ --CH₂--CH₂ --CH₂ --, --CH₂ --CH₂ --CH₂ --CH₂ --CH₂ CH₂ --, --CH₂ --NH--CH₂--CH₂ --CH₂ --, CH₂ --CH₂ --NH--CO--CH₂ --CH₂ --, --CH₂--NH--NH--CO--CH₂ --, and --CH₂ --O--CH₂ --NH--CO--CH₂ --. The carbons,nitrogens, and oxygens may be located at any position in these exemplarycompounds and, as above, the hydrogens may be independently substitutedwith other groups, provided that only one radical X is sp² or sp carbon,amide or imide nitrogen.

Compounds of the type described above and represented by Formula I canbe prepared by procedures described in the examples. One of ordinaryskill, upon reading the examples and examining the structure representedby Formula I, will be able to synthesize the plurality of compoundsrepresented by Formula I by substituting appropriate reagents in thereaction schemes provided.

Examples of bifunctional compounds of the instant invention suitable forconjugation to a protein comprise those compounds represented byFormulae II-XIV. ##STR12## Wherein the symbols T, R₀, R₁, R₆, R₁₁, R₁₂,R₁₃, and n are as described for formula I above, with the proviso that:

(a) each compound comprises at least one substituent R₁₅ --Z and onesubstituent R₁₆ ;

(b) when R₆ is R₁₆ or R₁₅ --Z, each R₁ group attached to a carbon atomadjacent to --N--R₆ is hydrogen;

(c) when R₁₁ Is R₁₆ or R₁₅ --Z, each R₁ group attached to a carbon atomadjacent to --N--R₁₁ is hydrogen.

Compounds of the type represented by Formulae II-IV includediamidodimercapto chelates, representative examples of which includeS-1-ethoxyethylmercapto-acetyl-γ-(2,3,5,6,-tetrafluorophenoxy)-L-glutamyl-α-S-acetamidomethyl-L-cysteinyl-6-amino-6-deoxy-D-gluconicacid. Compounds of the type represented by Formulae VI-IXinclude-monoamino monoamido mercaptides, representative examples ofwhich include4-N-(S-1-ethoxyethylmercaptoacetyl)-5-N'-(isothiocyanatophenethyl)-N'-.beta.-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoyl-6'-amino-6'-deoxy-D-gluconicacid. Compounds of the type represented by Formula X include diaminomercaptides, representative examples of which include4-N-methyl-N-β-(S-1-ethoxyethyl)mercaptoethyl-5-N'-(p-isothiocyanatophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoyl-6'-amino-6'-deoxy-D-gluconicacid. Preferred compounds of the type represented by Formulae XI-XIV areN₂ S₂ chelates containing an aromatic bridge, representative examples ofwhich include3-(β-1-ethoxyethyl)-acetamido-4-N-(β-1-ethoxyethyl)ethyl-N-(N',N'-dicarboxymethyl)-aminoethylphenylisothiocyanate. The chemical structure of one preferred compoundof the instant invention is represented by Formula XV: ##STR13## whereinZ represents an active ester group or an isothiocyanate group.

Chelating compounds of the type represented by Formulae I-XV are usefulfor radiolabeling proteins. Generally, two approaches may be employed toradiolabel proteins with the bifunctional chelating agents describedherein. The first method is referred to as the post-form approach and isdepicted in Equations (3) through (5) below. The method of radiolabelinga protein such as an antibody, according to this approach, consists offirst reacting the bifunctional chelating compound with the antibody,thereby forming a chelating compound-antibody conjugate, then admixingthe conjugate with the radionuclide metal under conditions suitable forforming a radionuclide metal-chelate-antibody complex in which theradiometal is complexed to the first weak binding site of the chelate.This complex is then incubated at about 37° C. or below, causingintramolecular rearrangement of the complex such that the metal istransferred to the thermodynamically stable second site, therebyproducing a stable radionuclide metal-chelate bound to an antibody.##STR14##

One advantage of employing compounds of the instant invention when usedin the post-form approach is that high yields of a thermodynamicallystable chelated radionuclide metal can be achieved under conditions thatpreserve biological activity of the protein. The observation that thestable chelate can be formed at temperatures less than 37° C. greatlyenhances the preservation of biological activity of the antibody byavoiding denaturation and aggregation. Prior-art methods for formingthermodynamically stable metal-chelate complexes conjugated to proteinsoften require the addition of a weak or labile chelating agent tofacilitate transfer of the radiometal to the chelate-antibody complex.Even when this strategy is employed, heating to from 50° to 75° C. ormore is required to transfer the metal to the chelate antibody complex,resulting in significant loss of biological activity of the antibody.

Another advantage of employing compounds of the instant invention, isthat less nonspecific binding of the radionuclide metal to the proteinitself occurs. Nonspecific binding is undesirable because theradionuclide metal may attach to a weak radionuclide metal chelatingsite on the antibody per se resulting in loss of the label duringadministration to the patient. In the instant invention, the firstcomplexing site of the chelating compound "out-competes" nonspecificbinding sites for the radionuclide metal on the antibody. The complex soformed then transfers the radiometal to the strong chelating site(during incubation at room temperature or with the application of heat)obviating the problem of nonspecific binding.

Pre-Form Approach

The bifunctional chelating compounds of the instant invention are alsosuitable for use for radiolabeling proteins according to the pre-formapproach. This approach is represented by Formulae 6, 7, and 8 below.The method for radiolabeling proteins, such as antibodies according tothis approach, consists of first reacting the radionuclide metal withthe chelating compound of the instant invention under conditions wherethe radionuclide metal rapidly forms a complex at the first weak bindingsite of the chelating compound. The conditions necessary to form thiscomplex are essentially the same as those described above for thepost-form approach. The complex so formed may then be incubated at theappropriate temperature, causing an intramolecular rearrangement suchthat the radionuclide metal ultimately resides at the second site of thechelating compound, thereby forming a stable radionuclide metal-chelate.This stable radionuclide metal-chelate is then admixed with the antibodyunder conditions suitable for conjugation, resulting in athermodynamically stable radionuclide metal-chelate-antibody conjugate.##STR15##

One advantage of employing bifunctional chelating compounds of theinstant invention according to the pre-formed approach is that thestable radionuclide metal-chelate complex can be formed at a lowertemperature compared to most prior art compounds. This obviates thenecessity of heating the metal chelate reaction mixture to elevatedtemperatures (e.g., on the order of 75° C.). This is particularlyadvantageous in that heating may decompose reactive conjugation groups,such as active esters; Michael-type acceptors such as maleimides;activated halides; and isothiocyanates. Incubation at temperaturesbetween room temperature and about 37° C. is more convenient in hospitalor clinical laboratories. In addition, compounds of the presentinvention exhibit good yields in the radiolabeling reaction, presumablybecause the first site and the second site are physically attached toeach other.

The specific methods for binding a radionuclide metal to the antibodyvia either the pre-formed or post-formed approach described above willdepend upon the particular radiometal selected and its normal valence.Suitable procedures are known to those skilled in the art.

As discussed above, pertechnetate and perrhenate are contacted with areducing agent to generate ^(99m) Tc and ¹⁸⁸ Re, respectively, in anoxidation state suitable for complex formation. The pertechnetate orperrhenate may be contacted with the reducing agent in a reactionmixture which also comprises a chelating compound so that theradionuclide metal is quickly bound in the form of a complex before theradionuclide metal can return to its oxidized state. An alternativeprocedure which may be used when a particular chelating compound (or aprotein attached thereto) may be adversely affected by contact with thereducing agent involves preparation of an initial exchange complex. Theexchange complex is prepared by reducing pertechnetate or perrhenate inthe presence of a separate complexing molecule (e.g., stannous gluconatefor ^(99m) Tc or stannous citrate for ¹⁸⁸ Re) to form ^(99m)Tc-gluconate or ¹⁸⁸ Re-citrate exchange complexes, respectively. Theexchange complex is contacted with a chelating compound of theinvention, whereupon the radionuclide metal is transferred to the firstsite, and ultimately becomes chelated at the second site.

The subject radionuclide metal-chelate-protein conjugates will beadministered to the mammalian host, normally by injection,intravenously, intraarterially, peritoneally, intratumorally, or thelike, depending upon the particular site at which the radionuclide metalis desired. Generally, from about 0.1 to 2 mL will be injected into ahost for diagnostic purposes, depending upon the size of the host, withabout 0.001 to 50 uCi/kg of host. For human hosts the dosage willusually be about 10-50 mCi/70 kg host, more usually about 25-35 mCi/70kg host. When the radionuclide metal-chelate-protein conjugates are tobe injected into the bloodstream of a human, the total volume injectedmay be larger; e.g., 20 to 30 mL administered by intravenous infusion.For lower mammals (e.g., mice), about 1 to 50 uCi is administered forbiodistribution studies, while up to or greater than 500 uCi isadministered for imaging studies. After administration of theradionuclide metal-chelate-protein conjugate, depending upon itspurpose, the host may be treated in various ways for detection ortherapy.

The diagnostic uses of the radionuclide metal-chelate-protein conjugatesof the invention thus provide a method for detecting the presence orabsence of a particular target site within a human or mammalian host. Ingeneral, such a conjugate is administered to the host, and thebiodistribution of the ^(99m) Tc is detected after waiting apredetermined length of time to allow accumulation of the compound atthe target site. The diagnostic procedures may vary according to theprotein component of the conjugate and other factors.

Technetium-99m (^(99m) Tc) has a physical half-life of 6 hours. Wholeimmunoglobulins have a biological half-life in serum of approximately 24hours (wide range), and thus the clearance of ^(99m) Tc-labeled antibodyfrom the circulation is slow compared to the physical half-life of^(99m) Tc. A ^(99m) Tc-labeled F(ab')₂ fragment has a shortercirculation time (T 1/2 9-20 hours) than whole immunoglobulin, which ismore compatible with tumor localization and background clearance for the^(99m) Tc-labeled antibody fragment to provide sufficienttumor:background ratios to image lesions successfully. Smaller fragmentssuch as Fab', Fab, and F_(v) have shorter circulation times (T 1/2 lessthan 180 minutes) that are more compatible with the physical T 1/2 of^(99m) Tc and are thus preferred for imaging applications. Choice ofmolecular species of antibody for imaging with other radionuclide metalswill similarly depend on the relationship of the physical half-life ofthe radionuclide metals and the circulation time of the molecularspecies of antibody.

Choice of molecular species of antibody for therapy applications ofradionuclide metals is more complex. In addition to physical andbiological half-lives, residence time of the labeled antibody in thetumor, energy of the emission, and contribution of total body tospecific organ dose are critical issues that dictate the optimal size ofantibody or fragment. With monoclonal antibodies, the particularantibody will also be a factor influencing the choice.

¹⁸⁸ Re has a 17-hour physical half-life, for which F(ab')₂ and Fabantibody fragments have suitable serum half-lives for tumor localizationand background clearance. The ¹⁸⁸ Re-labeled Fab would be expected tocause less toxicity to the bone marrow, but it will usually have ashorter residence time in tumor due to the lower affinity of univalentcompared to bivalent fragments. A ¹⁸⁸ Re-labeled Fab fragment with asuitably high affinity to maximize tumor residence of the deliveredcounts is especially useful.

¹⁸⁶ Re has a 3.67 day physical half-life. It can be used with wholeantibody or F(ab')₂ or smaller fragments thereof. Because the betaenergies are decreased compared to ¹⁸⁸ Re, the labeled antibody willneed to have a longer residence time in the tumor.

¹⁰⁹ Pd has a half-life of 14 hours. Antibody fragments, as opposed towhole antibodies, are expected to generally be most suitable forradiolabeling in accordance with the invention.

²¹² Pb has a physical half-life of 10.8 hours. Fab', Fab, or F_(v)fragments radiolabeled with ²¹² Pb would provide the greatest tumoruptake and background clearance in that period. ²¹² Pb decays to ²¹² Bi,which has an alpha emission with a physical half-life of 60 minutes. ²¹²Bi itself is not a feasible label unless compartmental administration(e.g., intraperitoneal) is used. ²¹² Pb will transmute to ²¹² Bi insitu, and it is necessary to use a ligand that can withstand the recoilfrom B-decay.

⁶⁷ Cu has a physical half-life of 2.44 days. In general, wholeantibodies or F(ab')₂ fragments thereof are most suitable forradiolabeling with this isotope for therapeutic use.

Delivery of the radionuclide metal-chelate-antibody conjugate may occurintravenously or by intraperitoneal, intralymphatic, intrathecal, orother intracavitary routes. Advantageously, an unlabeled(nonradiolabeled) antibody reactive with the same epitope as aradiolabeled antibody of the invention is administered prior toadministration of the radiolabeled antibody, as described in copendingU.S. patent application Ser. No. 917,176, filed Oct. 9, 1986, entitled"Methods for Improved Targeting of Antibody, Antibody Fragments, andConjugates Thereof." The nonradiolabeled antibody functions as an"unlabeled specific blocker" to decrease binding of thelater-administered radiolabeled antibody to cross-reactive sites thatmay be present on nontarget tissue. Blocking of such cross-reactivesites is important because antibodies generally have somecross-reactivity with tissues other than a particular target tissue. Inthe case of antibodies directed again tumor-specific antigens, forexample, virtually all such antibodies have some cross-reactivity withnormal (i.e., nontumor) tissues, with the exception of anti-idiotypes toB-cell lymphoma.

The unlabeled (cold) specific blocker protein advantageously isadministered from about 5 minutes to about 48 hours, most preferablyfrom about 5 minutes to about 30 minutes, prior to administration of theradionuclide metal-chelate-protein conjugate. The length of time mayvary according to such factors as the nature of the antibody and therelative accessibility of target sites versus cross-reactive bindingsites. The unlabeled specific blocker and the radionuclidemetal-chelate-antibody conjugate may be the same (except for theradiolabeling) or different, as long as both recognize the same epitope.In one embodiment of the invention, the unlabeled specific blocker is abivalent form of an antibody (e.g., a whole antibody or a F(ab')₂fragment thereof) and the radiolabeled polypeptide is a monovalentfragment of the same antibody (e.g., a Fab', Fab, or F_(v) fragment).Use of a bivalent form of an antibody as the cold specific blocker and amonovalent form for the radiolabeled antibody has the advantage ofminimizing displacement of the blocker from cross-reactive sites by thelater administered radiolabeled antibody due to the greater affinity ofthe bivalent form. The unlabeled specific blocker polypeptide isadministered in an amount effective in binding with (blocking) at leasta portion of the cross-reactive binding sites in a patient. Thus,binding of a radiolabeled polypeptide to cross-reactive binding sitesmay be reduced, thereby improving diagnostic imaging of target sitesand, in general, reducing somewhat the amount of radiolabeled antibodyto be administered. The amount may vary according to such factors as thesize of the patient and the nature of the polypeptide. In general, about5 mg or more of the unlabeled specific blocker is administered to ahuman.

Advantageously, a second antibody, termed an "irrelevant" antibody, isalso administered to a patient prior to administration of theradiolabeled polypeptide, as described in U.S. patent application Ser.No. 917,176. The irrelevant antibody is an antibody that does not bindto sites within the patient by a specific (e.g., antigen-binding)mechanism but which may bind to target and nontarget sites throughnonspecific mechanisms (e.g., adsorption or binding of the Fc portion ofthe irrelevant antibody to Fe receptors on cells in thereticuloendotheial system). The irrelevant antibody blocks certainnontarget sites in a patient and thus decreases nonspecific binding ofthe radiolabeled polypeptide to these nontarget sites. Diagnosticimaging of target sites thus may be improved, and the amount ofradiolabeled antibody to be administered may be somewhat reduced. Forexample, prior administrations of an irrelevant antibody that is notspecific for any human tissues, as far as is known, effectively reducedthe nonspecific uptake of whole and F(ab')₂ radiolabeled antibody intoliver and spleen in human patients.

The irrelevant antibody advantageously is administered from 5 minutes to48 hours, most preferably from 15 minutes to one hour, prior toadministration of the radiolabeled polypeptide. The length of time mayvary according to such factors as the nature of the antibody. Manysuitable antibodies that may be used as the irrelevant antibody areknown. For example, there are many known antibodies that are notspecific for any human tissues that may be used as the irrelevantantibody. In one embodiment of the invention, a murine monoclonalantibody to a B-cell lymphoma idiotype (i.e., specific only for thelymphoma cells of one individual human) is administered as theirrelevant polypeptide. In one embodiment of the invention, theirrelevant polypeptide is a whole antibody or a F(ab')₂ fragmentthereof. The irrelevant polypeptide is administered in an amounteffective in blocking at least a portion of the sites at whichnonspecific binding (i.e., binding through nonspecific mechanisms) ofthe radiolabeled polypeptide occurs in the absence of the irrelevantpolypeptide. The amount may vary according to such factors as the natureof the polypeptides and the size of the patient. In general, about 15 mgor more (preferably less than 200 mg) of the irrelevant antibody isadministered.

The instant invention also includes a kit for producing radionuclidemetal-chelate-protein conjugates comprising a bifunctional chelatingcompound of the instant invention, and a protein to be radiolabeled. Theprotein may be any of the above-described proteins which, inradiolabeled form, have diagnostic or therapeutic applications. In oneembodiment of the invention, the protein of the instant kit is anantibody, preferably a monoclonal antibody such as a monoclonal antibodyspecific for cancer cells. In an alternative embodiment of the presentinvention, the kit includes a protein having a chelating compound of theinvention conjugated thereto.

Reagents useful in reactions to radiolabel the chelating compound with aradionuclide metal and to conjugate the chelate compound to thepolypeptide according to either the pre-form or post-form approach mayalso be included. Such kits also may comprise a means for purifying theradiolabeled polypeptide from the reaction mixture, as well as specificinstructions for producing the radiolabeled polypeptide using the kitcomponents. Such kits generally will be used in hospitals, clinics, orother medical facilities. Since such facilities generally have readyaccess on a daily basis to radionuclide metals, such as isotopes oftechnetium, and since isotopes of rhenium, lead, bismuth, palladium, andcopper may be prepared as described above, inclusion of the radionuclidemetal in the kit is optional. Exclusion of the radionuclide metalpermits storage of the kit, whereas kits containing the radionuclidemetal (either as a separate component or as the radiolabeled chelatecompound) would have to be used within a narrow time frame (depending onthe half-life of the particular isotope); otherwise, radioactive decayof the radioisotope would diminish the effectiveness of the diagnosticor therapeutic technique for which the radiolabeled protein is used. For¹⁸⁶ Re, on-site radiolabeling would avoid radiolytic degradation of thelabeled antibody due to the beta particle emission.

The kits may be diagnostic or therapeutic kits, depending on whichradioisotope is used for labeling the chelating agent. When theradionuclide metal is to be reduced to a lower oxidation state (e.g.,technetium and rhenium, as discussed above), the kits may additionallycomprise a reducing agent effective in reducing a particularradionuclide metal, to be chelated by the chelating compound, to anoxidation state at which a complex of the radionuclide metal-chelate maybe formed.

The bifunctional chelating compounds may be radiolabeled with aradionuclide metal prior to or after conjugation to a protein. A kitsuitable for use in the pre-formed approach preferably includes abifunctional chelating compound comprising sulfur-protecting groups anda protein in separate containers. A kit suitable for use in thepost-form approach may contain the bifunctional chelating compound andthe protein in separate containers, for conjugation at a later time, orthe chelating compound may already be conjugated to a protein. The term"separate containers" as used herein is meant to include not onlyseparate, individual containers (e.g., vials) but also physicallyseparate compartments within the same container. Bifunctional chelatingcompounds of the instant invention are preferably already conjugated toa protein.

In accordance with one embodiment of the invention, a diagnostic kitsuitable for use according to the post-form approach comprises thefollowing reagents (in separate containers unless otherwise noted),presented in the general order of use.

1. A reducing agent effective in reducing pertechnetate (^(99m) TcO₄) toa lower oxidation state at a neutral to acidic pH so that atechnetium-chelate complex can be formed. Many suitable reducing agentsare known, including but not limited to, stannous ion, (e.g., in theform of stannous salts, such as stannous chloride or stannous fluoride),metallic tin, formamidine sulfinic acid, ferric chloride, ferroussulfate, ferrous ascorbate, and alkali salts of borohydride. Preferredreducing agents are stannous salts.

2. A chelating compound of the invention conjugated to a protein orfragment thereof specific for the desired target organ, tissue, antigen,or other target site within a mammalian body, as discussed above.

3. Means for purifying the desired radionuclide metal-chelate-proteinconjugate from the reaction mixture. Any suitable known proteinpurification technique may be used that effectively separates thedesired radiolabeled protein conjugate from other compounds in thereaction mixture. The purification step may, for example, separate thedesired conjugate from impurities due to differences in size or inelectrical charge. One suitable purification method involves columnchromatography, using, for example, an anion exchange column or a gelpermeation column. Good results are also achieved by columnchromatography using an anion exchange column, e.g., a quaternaryaminoethyl Sephadex (QAE-Sephadex) column or a diethylaminoethylSephadex (DEAE-Sephadex) column. Since virtually all the impurities tobe removed (e.g., sodium pertechnetate and technetium dioxide), arenegatively charged, they are substantially retained on the positivelycharged column. Purification thus may be accomplished by this one-stepcolumn procedure.

4. Additional reagents for use in the radiolabeling the protein-chelateconjugate (e.g., the buffers, alcohols, acidifying solutions, and othersuch reagents, as described below) are generally available in medicalfacilities and thus are optional components of the kit. However, thesereagents preferably are included in the kit to ensure that reagents ofsufficient purity and sterility are used because the resultingradionuclide metal-chelate-protein conjugates are to be administered tomammmals, including humans, for medical purposes.

5. Optionally, a container of a polypeptide to be administered innonradiolabeled form to a human or mammal is included in the kit. Thispolypeptide is reactive with essentially the same target site as thepolypeptide to be radiolabeled and reduces binding of the radiolabeledpolypeptide to cross-reactive binding sites on nontarget tissues. Thetwo polypeptides may be the same, or the polypeptide to be radiolabeledmay, for example, be a fragment of the polypeptide that is to beadministered In nonradiolabeled form. The latter polypeptide isadministered as an unlabeled specific blocker (prior to administrationof the radiolabeled polypeptide) in an amount effective in improvingdiagnostic imaging of the desired target sites (e.g., tumors), asdescribed above.

6. Optionally, the kit also comprises a container of a polypeptide thatdoes not bind through specific mechanisms to sites within the human ormammal to which the radiolabeled polypeptide is to be administered. Thispolypeptide is administered as an "irrelevant" polypeptide (prior toadministration of the radiolabeled polypeptide) in an amount effectivein decreasing nonspecific uptake of certain radiolabeled polypeptides,as described above.

The following specific examples are intended to illustrate more fullythe nature of the present Invention without acting In any way to limitits scope.

EXAMPLE I Synthesis of a diamidodimercapto bifunctional anchimericchelateN-(S-1-ethoxyethylmercaptoacetyl)-γ-(2,3,5,6,-tetrafluorophenoxy)L-glutamyl-α-S-acetamidomethyl-L-cysteinyl-6-amino-6-deoxy-D-gluconicacid

The following synthesis is best understood by referring to FIGS. 1a and1b.

N-(t-butoxycarbonyl)-γ-(t-butoxy)-L-glutamyl-S-acetamidomethyl-L-cysteine3: A solution of S-(acetamidomethyl)-L-cysteine (2 mmol) 1 in 10 mL ofanhydrous dimethylformamide containing 4-dimethylaminopyridine (2 mmol)is cooled in an salt-ice bath to 0°-5° C. To this solution is addedpreviously cooled isobutylchloroformate (2 mmol), and the solution isincubated at this temperature for another 30 minutes. A solution ofN-(t-butoxycarbonyl)-L-glutamic acid-γ-butyl ester 2 (2 mmol) in 5 mL ofdimethylformamide is added at such a rate that the temperature of thereaction mixture does not exceed 0° C.

The mixture is kept at this temperature for another 1 hour and allowedto come to room temperature. The solution is extracted with 2×20 mL ofmethylene chloride and dried over anhydrous sodium sulfate. Evaporationand crystallization give the dipeptide derivative 3.

N-(t-butoxycarbonyl)-γ-(t-butoxy)-L-glutamyl-S-acetamidomethyl-L-cysteinyl-6-amino-6-deoxy-D-gluconicacid 5:

(i) A solution of 3 (1 mmol) in 10 mL of anhydrous tetrahydrofuran isstirred with 1.1 mmol of N-hydroxysuccinimide and 1.1 mmol ofN,N'-dicyclohexylcarbodiimide. After stirring overnight the precipitateddicyclohexylurea is filtered and the filtrate is evaporated to dryness.The residue is dissolved in 20-25 mL of ethyl acetate and washed withwater, and the organic layer is dried over anhydrous sodium sulfate.Evaporation and crystallization give the succinimidate ester as a solid.

(ii) To a solution of the above ester (1 mmol) in a mixture of 1:1acetonitrile:water, 6-amino-6-deoxy-D-gluconic acid 4 (prepared from1,2-O-isopropylidine-α-D-glucopyranose according to the procedure of K.Kefurt et. al., Collection Czecholslov. Chem. Comm., 44, 2526 (1979)) isadded, and the mixture is stirred for 6 hours at room temperature. Theproduct 5 is isolated by evaporation followed by flash chromatographyover silica gel.

N-(t-butoxycarbonyl)-γ-(t-butoxy)-L-glutamyl-S-acetamidomethyl-L-cysteinyl-6-amino-6-deoxy-D-gluconicacid trimethylsilylethyl ester 6: To 5 mL of a solution containing 1mmol of the above acid 5, anhydrous dimethylformamide, and 1 mmol ofN-methylmorpholine maintained at 0°-5° C., 1 mmol ofisobutylchloroformate is added, and the mixture is kept at thistemperature for 20 minutes. To this solution is added a solution of 1mmol of trimethylsilylethanol in 2 mL of anhydrous dimethylformamide(previously cooled to 0°-5° C.), and the solution is stirred for 1 hourat this temperature and allowed to come to room temperature. The solventis removed in vacuo and the residue is dissolved in 20 mL of ethylacetate and washed with water. The organic layer is dried and evaporatedto give 6.

L-glutamyl-α-S-acetamidomethyl-L-cysteinyl-6-amino-6-deoxy-D-gluconicacid trimethylsilylethyl ester 7: The above ester (1 mmol) is dissolvedin 5 mmol of anhydrous trifluoroacetic acid (previously cooled to 0° C.)and stirred for 3 hours. The solution is allowed to come to roomtemperature and is evaporated in vacuo. Trituration with ether yieldsthe amino acid 7 as the trifluoroacetate salt.

N-(S-1-Ethoxyethylmercaptoacetyl)-L-glutamyl-α-S-acetamidomethyl-L-cysteinyl-6-amino-6-deoxy-D-gluconicacid trimethylsilylethyl ester 9: Reagent 8 is prepared by firstpreparing S-(1-ethoxyethyl)mercaptoacetic acid 16 (as described below).

The S-(1-ethoxyethyl)mercaptoacetic acid (5.76 g, 35.1 mmol) is combinedwith N-hydroxysuccinimide (4.85 g, 42.1 mmol) in 100 mL of anhydrousTHF. To this is added a solution of 1,3-dicyclohexylcarbodiimide (8.70g, 42.1 mmol) in 65 mL of anhydrous THF. The mixture is stirred at roomtemperature for 2 hours, or until TLC analysis indicates completeformation of the succinimidyl ester. The mixture is then filtered, andthe filtrate is concentrated in vacuo to a viscous residue. The residueis dissolved in ethyl acetate, washed with water, brine, and dried(MgSO₄). Removal of the solvent leaves the crude succinimidyl ester asan oil, which is further purified by flash chromatography on silica gel,using ethyl acetate:hexanes as the column eluant, to give 5.1 g ofS-1-ethoxyethylmercaptoacetic acid succinimidyl ester 8 as a colorlessoil.

To a solution of 7 (1 mmol) in 10 mL of anhydrous dimethylformamidecontaining 1 mmol of triethylamine, 1 mmol ofS-(1-ethoxyethyl)mercaptoacetic acid succinimidate ester 8 is added andthe solution is stirred for three hours at ambient temperature. Thesolvent is removed in vacuo, and the residue is dissolved in ethylacetate and washed with water. The organic layer is dried andevaporated, and the product is purified by flash chromatography to givethe product 9.

N-(S-1-Ethoxyethylmercaptoacetyl)-γ-(2,3,5,6,-tetrafluorophenoxy)L-glutamyl-α-S-acetamidomethyl-L-cysteinyl-6-amino-6-deoxy-D-gluconicacid trimethylsilylethyl ester 10: To a solution of 9 (1 mmol) in 20 mLof anhydrous tetrahydrofuran, 2,3,5,6-tetrafluorophenol andN,N'-dicyclohexylcarbodiimide is added, and the solution is stirred for10-12 hours at room temperature. The precipitated dicyclohexylurea isfiltered, and the solvent is removed by evaporation under reducedpressure. The residue is dissolved in 20-30 mL of ethyl acetate andwashed with water. The organic layer is dried and evaporated, and theproduct 10 is isolated by flash chromatography over silica gel.

N-(S-1-Ethoxyethylmercaptoacetyl-γ-(2,3,5,6-tetrafluorophenoxy)L-glutamyl-α-S-acetamidomethyl-L-cysteinyl-6-amino-6-deoxy-D-gluconicacid 11: To a solution of 1 mmol of 10 in 10 mL of anhydroustetrahydrofuran, 3 mL of 1M tetra-n-butylammonium fluoride is added, andthe solution is stirred for 6-8 hours at ambient temperature. Thesolvent is removed by evaporation, and the product 11 is isolated byflash chromatography.

EXAMPLE II

Synthesis of an amino amido dimercapto bifunctional anchimeric chelate4-N-(S-1-ethoxyethylmercaptoacetyl)-5-N'-(p-isothiocyanatophenethyl)-N'-.beta.-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoyl-6'-amino-6'-deoxy-D-gluconicacid

The following synthesis is best understood by referring to FIGS. 2a and2b.

N-t-Butoxycarbonyl-β-carbomethoxyethyl aziridine 15:

(i) N-t-butoxycarbonyl-L-glutamic acid-γ-methyl ester 12 is preparedfrom L-glutamic acid-γ-methyl ester according to the procedure of R. K.Olsen and T. Emery., J. Org. Chem., 49:3527 (1984).

(ii) A 1.0M solution of borane:THF (0.68 mL, 0.68 mmol) was added to asolution of 12 (143 mg. 0.55 mmol) in anhydrous tetrahydrofuran (0.68mL). The reaction solution was stirred at ambient temperature for onehour and then quenched by the addition of 10 mL of methanol. Thereaction solution was then evaporated to give an oil (160 mg). The oilwas dissolved in 70 mL of ethyl acetate and washed with saturated NaHCO₃(2×30 mL). The organic layer was dried over anhydrous MgSO₄ andevaporated in vacuo to give 13 as a colorless oil (120 mg, 88%).

(iii) p-Toluenesufonyl chloride (0.85 g, 4.45 mmol) was added to an icecold (0°-5° C.) solution of 13 (1.00 g, 4.05 mmol) in pyridine (8 mL).The reaction solution was stirred at this temperature overnight. Thereaction solution was diluted with methylene chloride (80 mL) and washedwith pH 4.0 buffer (3×70 mL), then with saturated bicarbonate (40 mL).The organic extract was repeatedly evaporated from toluene (to azeotropethe pyridine), giving the tosylate 14 as a brown viscous oil that wasused in the next step without further purification.

(iv) A solution of the tosylate 14 (1.38 g) in anhydrousdimethylformamide (3.0 mL) was added to a suspension of NaH (95 mg, 3.78mmol) in DMF (1.5 mL). The reaction mixture was stirred for 1 hour,diluted with water (40 mL), and extracted with methylene chloride (3×40mL). The combined methylene chloride extracts were dried (MgSO₄) andevaporated to give a yellow oil (0.66 g). The oil was purified by flashchromatography over silica gel (1:1 ethyl acetate:hexanes) to give 15 asa pale yellow oil.

(S-1-Ethoxyethyl)-N-(p-nitrophenethyl)mercaptoethylamine 19:

(i) S-(1-ethoxyethyl)mercaptoacetic acid 16 was prepared according tothe following procedure: A solution of mercaptoacetic acid (17.4 mL, 250mmol) in 125 mL of dichloromethane containing p-toluenesulfonic acidmonohydrate (0.24 g, 1.26 mmol) was cooled to -18° to -25° C. withstirring. Ethyl vinyl ether (23.9 mL, 250 mmol) in 125 mL ofdichloromethane was added dropwise to the cold solution over a period of90 minutes. The stirring was continued for an additional 30 minutes,with the temperature maintained in the -18° to -25° C. range. Then 200mL of pH 7 phosphate buffer was added, and the reaction mixture wasallowed to warm with stirring for 10 to 15 minutes. The mixture was thenpoured into a flask containing 900 mL of ethyl acetate and 200 mL ofwater. Layers were separated, and the aqueous portion extracted twicewith ethyl acetate. The organic layers were combined, washed with brine,and dried (MgSO₄). Removal of the solvent left 31.4 g ofS-(1-ethoxyethyl)mercaptoacetic acid 16 as a colorless oil (77% yield):¹ H NMR (CDCl₃) 1.15(t,J=7.0 Hz,3H), 1.52(d,J=6.4 Hz,3H), 3.36(s,2H),3.60(m,2H), 4.84(q,J=6.4 Hz, 1H), 11.65(s, 1H). The material was usedwithout further purification.

(ii) To a solution of 3.07 g of 16 in 65 mL of anhydrous tetrahydrofuran(kept at 0° C.) 93.5 mL of borane:THF complex (1M) was added slowly. Themixture was stirred for 7.5 hours in a nitrogen atmosphere at 0° C.Approximately 500 mL of water was added slowly to the reaction mixtureand stirred for 15 minutes. The mixture was concentrated under vacuum at40°-50° C. and the aqueous residue was extracted with ethyl acetate. Theethyl acetate layer was washed with 50 mL of 10% Na₂ CO₃. The aqueouslayer was washed again with 2×25 mL of ethyl acetate. The combinedorganic layers were washed with brine, dried, filtered, and evaporatedto give 1.7 g of an oil. The oil was purified in an HPLC to give 1.34 gof 17 as a colorless oil.

(iii) To a solution of 17 (1 mmol) in 5 mL of methylene chloridecontaining 1 mmol of triethylamine maintained at 0° C. was addedmethanesulfonyl chloride (1 mmol) and the solution stirred for one hourat this temperature. The mesylate 18 was unstable and hence theamidation was carried out without isolation.

To the above solution 1.1 mmol of p-nitrophenethylamine is added, andthe solution is stirred at this temperature for two hours and allowed tocome to ambient temperature. After stirring overnight at roomtemperature, the solution is diluted with saturated bicarbonate,extracted with methylene chloride. The combined organic extracts arewashed with brine, dried over anhydrous MgSO₄, and evaporated to give 19as a viscous oil, which is purified by flash chromatography over silicagel.

4-N-(t-butoxycarbonyl)-5-N'-(p-nitrophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoicacid 21:

(i) To a solution of N-t-butoxycarbonyl-S-carbomethoxyethyl aziridine 15(1 mmol) in anhydrous tetrahydrofuran (5 mL), is addedS-ethoxyethyl-N-(p-nitrophenethyl)mercaptoethylamine 19, and the mixtureis refluxed for 6-8 hours. The solvent is removed in vacuo, and theproduct 204-N-(t-butoxycarbonyl)-5-N'-(p-nitrophenethyl)-N'-β-(S-ethoxyethyl)-mercaptoethyl-diaminopentanoicacid methyl ester is used in the next step without further purification.

(ii) To a solution of the above compound (1 mmol in 5 mL of methanol),0.7 mL of 2N NaOH is added, and the mixture is stirred for overnight atroom temperature. The solution Is concentrated to a small volume andacidified to pH 2-3 with 0.5N HCl. The precipitated solid 21 iscollected by filtration and purified by flash chromatography over silicagel and crystallization.

4-N-(t-butoxycarbonyl)-5-N'-(p-nitrophenethyl)-N'-β-(S-1-ethoxyethyl)-mercaptoethyl-diaminopentanoyl-6'-amino-6'-deoxy-D-gluconicacid 23:

(i) A solution of 21 (1 mmol) in 15 mL of anhydrous tetrahydrofuran isstirred with 1.1 mmol of N-hydroxysuccinimide and 1.1 mmol ofN,N'-dicyclohexylcarbodiimide. After stirring overnight at roomtemperature, the precipitated solid is filtered and the solutionevaporated. The residue is dissolved in 2×20-25 mL of ethyl acetate andwashed with water. The organic layer is dried over anhydrous Na₂ SO₄ andevaporated. The product obtained 22 crystallized form ethyl acetate.

(ii) The above succinimidate 22 ester (1 mmol) is dissolved in 20 mL ofacetonitrile:water (4:1) to which 1 mmol of 6-amino-6-deoxy-D-gluconicacid 4 is added in one lot, and the mixture is stirred for three hoursat room temperature. The solvent is removed in vacuo, and the residue ischromatographed to give the product 23 as a solid.

4-N-(t-butoxycarbonyl)-5-N'-(p-nitrophenethyl)-N'-β-(S-1-ethoxyethyl)-mercaptoethyl-diaminopentanoyl-2',3',4',5'-tetra-O-t-butyldimethylsilyl-6'-amino-6'-deoxy-D-gluconicacid 24: To a solution of 23 (1 mmol) in anhydrous tetrahydrofurancontaining triethylamine (5 mmol), t-butyldimethylsilyl chloride isadded. The mixture is stirred for 8-10 hours at room temperature. Thesolvent is evaporated, and the residue is crystallized and dissolved inethyl acetate (20-30 mL), then washed with brine, dried with anhydrousNa₂ SO₄, and evaporated to obtain the t-butyldimethylsilyl derivative24.

4-N-(t-butoxycarbonyl)-5-N'-(p-isothiocyanatophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoyl-2',3',4',5'-tetra-O-t-butyldimethylsilyl-6'-amino-6'-deoxy-D-gluconic acid 25:

(i) A solution of 24 (1 mmol) in 50 mL of ethanol containing 100 mg ofsulfided Pd/C (5%) is shaken in a Paar hydrogenator at a pressure of 60psi for 15-20 hours. The catalyst is removed by filtration throughcelite, and the filtrate is evaporated to give the amino compound4-N-(t-butoxycarbonyl)-5-N'-(p-aminophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoyl-2',3',4',5'-tetra-O-t-butyldimethylsily-6'-amino-6'-deoxy-D-gluconicacid as an oil, which is used in the next step without furtherpurification.

(ii) To a solution of the above amino compound in 25 mL of methylenechloride is added thiocarbonyldiimidazole (1.1 mmol) and the mixture isstirred overnight at room temperature. The solution is diluted withanother 20 mL of methylene chloride and washed with water. Evaporationand crystallization give the isothiocyanate 25 as a solid.

4-N-(S-1-ethoxyethylmercaptoacetyl)-5-N'-(p-isothiocyanatophenethyl)-N'-.beta.-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoyl-6'-amino-6'-deoxy-D-gluconicacid 26

(i) A solution of 25 (1 mmol) is stirred with 5 mL of anhydroustrifluoroacetic acid for 3-4 hours at room temperature. One mL of wateris added to the reaction mixture and the mixture is evaporated to givean oily product. This residue is triturated with organic solvent to givethe requisite intermediate5-N-(p-isothiocyanatophenethyl)-N-β-(S-1-ethoxyethyl)mercaptoethyl-4,5-diaminopentanoyl-6'-amino-6'-deoxy-D-gluconicacid.

(ii) To a solution of the above compound (1 mmol) in 5 mL of anhydrousdimethylformamide containing an equimolar amount of triethylamine isadded 1.1 mmol of S-1-ethoxyethylmercaptoacetic acid succinimidate esterand the mixture is stirred for 6 hours at room temperature. The solventis removed in vacuo, and the residue containing the product 26 andN-hydroxysuccinimide is shaken with ethyl acetate to remove theunreacted N-hydroxysuccinimide. The filtered solid is purified bypreparative liquid chromatography to give a pure product 26.

EXAMPLE III Synthesis of a diamino dimercapto bifunctional anchimericchelate 4-N-methyl-N-β-(S-1-ethoxyethyl)mercaptoethyl-5-N'-(p-thiocyanatophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoyl-6'-amino-6'-deoxy-D-gluconicacid.

The following synthesis is best understood by referring to FIGS. 3a and3b.

N-Methyl-β-carbomethoxyethyl aziridine 31:

(i) To a solution of t-butoxycarbonyl-L-glutamic acid-γ-methyl ester 12(1 mmol) in 10 mL of anhydrous tetrahydrofuran, 1.1 mmol oftrimethylsilyl ethanol and 1.1 mmol of N,N'-dicyclohexylcarbodiimide isadded, and the solution is stirred overnight. The precipitated solid isfiltered, and the filtrate is evaporated. The residue is dissolved inethyl acetate and washed with water, dried with anhydrous MgSO₄,evaporated, and crystallized to give 27 N-t-butoxycarbonyl-L-glutamicacid-(α-trimethylsilylester)-γ-methyl ester.

(ii) To a solution of 27 (N-t-butoxycarbonyl-L-glutamicacid-(α-trimethylsilylester)-γ-methyl ester) (1 mmol) in 10 mL ofanhydrous methylformamide, 3 mmol of silver oxide is added, and themixture is kept at 37°-40° C. After 24 hours at room temperature, themixture is filtered and evaporated to dryness. The residue is dissolvedin CH₂ Cl₂, washed with water, dried, and evaporated to give 28(N-t-butoxycarbonyl-N-methyl-L-glutamicacid-[α-trimethylsilylester)-γ-methyl ester.

(iii) A solution of 28 (2 mmol) in 10 mL of anhydrous tetrahydrofuran isstirred with 5 mL of 1M tetra-n-butylammonium fluoride for six hours.The solvent is removed, and the residue is washed with water, dried, andevaporated to give N-t-butoxycarbonyl-N-methyl-L-glutamic acid-γ-methylester. This compound is converted to the product4-(t-butoxycarbonyl-methylamino)-4-(hydroxymethyl)-butanoic acid methylester 29, in a procedure similar to the one described for the conversionof 12 to 13.

(iv) A solution of 1 mmol of 29(4-(t-butoxycarbonyl-methylamino)-4-(hydroxymethyl)-butanoic acid methylester) is stirred with 5 mL of anhydrous trifluoroacetic acid for twohours and evaporated to dryness. The residue is triturated with ether togive the trifluoroacetate salt4-(methylamino)-4-(hydroxymethyl)-butanoic acid methyl ester 30.

(v) A solution of 30 (1 mmol) in anhydrous tetrahydrofuran is treatedwith pyridine SO₃ -complex (1.1 mmol) to give the O-sulfonate of 30.After TLC shows the disappearance of the starting material, the solutionis heated to boiling overnight to give the product 31(N-methyl-β-carbomethoxyethyl aziridine), which is recovered by removalof the solvent.

4-N-methyl-5-N'-(p-nitrophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyldiaminopentanoicacid methyl ester 32: This compound is prepared from 31 and 19(S-1-ethoxyethyl-N-(p-nitrophenethyl)mercaptoethylamine) in a proceduresimilar to the one described earlier for the preparation of 20 from 15and 19).

4-N-methyl-N-β-(S-1-ethoxyethyl)mercaptoethyl-5-N'-(p-nitrophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoicacid succinimidate ester 34:

(i) A mixture of 33 and 18 (S-ethoxyethyl-β-mercaptoethanol mesylate(EXAMPLE I) in equimolar amounts are heated in 10 mL of anhydroustetrahydrofuran for six hours. The solvent is removed in vacuo, and theresidue is dissolved in ethyl acetate and washed with water. The organiclayer is dried and evaporated to give 33,4-N-methyl-N-β-(S-1-ethoxyethyl)mercaptoethyl-5-N'-(p-nitrophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoicacid methyl ester.

(ii) The above methyl ester 33 is hydrolyzed to the free acid 34(4-N-methyl-N-β-(S-1-ethoxyethyl)mercaptoethyl-5-N'-(p-nitrophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyldiaminopentanoic acid) in a similar procedure described earlier for theconversion of 20 to 21 (see EXAMPLE I).

(iii) The free acid 34 is converted to the succinimidate ester 35(4-N-methyl-N-β-(S-1-ethoxyethyl)mercaptoethyl-5-N'-(p-nitrophenethyl)-N'-β-(S-ethoxyethyl)mercaptoethyldiaminopentanoic acid succinimidate ester) in a similar proceduredescribed for the conversion of 21 to 22 (see EXAMPLE I).

4-N-methyl-N-β-(S-1-ethoxyethyl)mercaptoethyl-5-N'-(p-nitrophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoyl-6'-amino-6'-deoxy-2',3',4',5'-tetra-O-t-butyldimethylsilyl-D-gluconicacid 36: Compound 35 is converted to 36 in two successive steps in aprocedure similar to the one described earlier for the conversion of 22to 24 (see EXAMPLE I).

4-N-methyl-N-β-(S-1-ethoxyethyl)mercaptoethyl-5-N'-(p-isothiocyanatophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoyl-6'-amino-6'-deoxy-2',3',4',5'-tetra-O-t-butyldimethylsilyl-D-gluconicacid 37: Compound 36 is converted to 37 in two successive steps in aprocedure similar to the one described earlier for the conversion of 24to 25 (see EXAMPLE I).

4-N-methyl-N-β-(S-1-ethoxyethyl)mercaptoethyl-5-N'-(p-isothiocyanatophenethyl)-N'-β-(S-1-ethoxyethyl)mercaptoethyl-diaminopentanoyl-6'-amino-6'-deoxy-D-gluconicacid 38.Deprotection of 37 to the target compound 38 is achieved bystirring the compound (1 mmol) in 10 mL acetonitrile containing 2 mL of1N hydrochloric acid for 2-3 hours. Evaporation of the solvent followedby purification by HPLC yields the product 38 in a pure state.

EXAMPLE IV Synthesis of a diamidodimercapto bifunctional anchimericchelate having an aromatic bridge3-(β-S-ethoxyethyl)-acetamido-4-N-(β-S-1-ethoxyethyl)ethyl-N-(N',N'-dicarboxymethyl)-aminoethylphenylisothiocyanate

The following synthesis is best understood by referring to FIGS. 4a and4b.

S-1-ethoxyethyl-N-β-dimethoxyethyl-mercaptoethylamine 39: A solution ofS-ethoxyethyl-O-mesyl-mercaptoethanol 18 (1 mmol) (see EXAMPLE II forprocedure) is stirred with aminoacetaldehyde dimethyl acetal at ambienttemperature for 5-6 hours. The solution is diluted with saturatedbicarbonate, extracted with methylene chloride. The combined organicextracts are washed with brine, dried over anhydrous sodium sulfate andevaporated in vacuo to give 39, which is purified by silica gel flashchromatography.

3-(β-S-1-ethoxyethyl)-acetamido-4-chloronitrobenzene 41: To a solutionof 2-chloro-4-nitroaniline 40 (1 mmol) in 10 mL of anhydrousdimethylformamide, 1.1 mmol of S-(1-ethoxyethyl)mercaptoacetic acidsuccinimidate ester 8 (see EXAMPLE II) is added and the mixture isstirred for 6-8 hours at ambient temperature. The solvent is removed invacuo, and water is added to the residue. The mixture is extracted withethyl acetate, washed with water, dried over anhydrous sodium sulfateand evaporated to give 41.

3-(β-S-1-ethoxyethyl)-acetamido-4-N-(β-S-1-ethoxyethyl)ethyl-N-(.beta.,β-dimethoxy)-ethylnitrobenzene 42: An equimolar amount of a mixture of3-(β-S-ethoxyethyl)-acetamido-4-chloronitrobenzene 41 andS-ethoxyethyl-N-β-dimethoxyethyl-mercaptoethylamine 39 heated in amixture of anhydrous dimethylformamide for a period of 4-5 hours. Thesolvent is removed in vacuo and the residue is suspended in water. Thesolution is rendered alkaline with 1N sodium bicarbonate and extractedwith methylene chloride. The organic layer is dried with anhydroussodium sulfate and evaporated to give the product 42.

3-(β-S-1-ethoxyethyl)-acetamido-4-N-(β-S-ethoxyethyl)ethyl-N-(N',N'-dicarbomethoxymethyl)aminoethylnitrobenzene 43:

(i) The above product (1 mmol) is treated with 5 mL of glacial aceticacid containing 1 mL 2N HCl and stirred at room temperature for 4-5hours and evaporated to dryness. The residue is dissolved in ethylacetate and washed with water. The organic layer is dried and evaporatedto give the formyl intermediate.

(ii) The formyl intermediate is dissolved in 10 mL of anhydroustetrahydrofuran and cooled to 0° C. To this a solution of an equimolaramount of iminodiacetic acid dimethyl ester in tetrahydrofuran is added.After stirring for 30 minutes at this temperature, 3-5 equivalents ofsodium cyanoborohydride is added in portions. The mixture is stirred foranother 2 hours and allowed to come to room temperature. The solvent isremoved in vacuo, and the residue is dissolved in ethyl acetate. Theorganic layer is washed with water, dried, evaporated, and purified bysilica gel chromatography to give product 43.

3-(β-S-ethoxyethyl)-acetamido-4-N-(β-S-ethoxyethyl)ethyl-N-(N',N'-dicarboxymethyl)aminoethylnitrobenzene 44: A solution of 43 (1 mmol) is dissolved in 2 mL ofethanol and 3 mL of 1N NaOH and stirred at room temperature overnight.The solution is evaporated under reduced pressure and redissolved in 5mL of water and acidified with 1N HCl to pH 3-4. The precipitated solidis filtered and dried to give 44.

3-(β-S-ethoxyethyl)-acetamido-4-N-(β-S-ethoxyethyl)ethyl-N-(N',N'-dicarboxymethyl)-aminoethylaniline 45: The reduction is carried according to the proceduredescribed in EXAMPLE II (reduction of 25 to the corresponding aminocompound).

3-(β-S-ethoxyethyl)-acetamido-4-N-(β-S-ethoxyethyl)ethyl-N-(N',N'-dicarboxymethyl)-aminoethylphenylisothiocyanate 46: The amino compound 45 is converted to theisothiocyanate in a procedure similar to that described in EXAMPLE IIusing thiocarbonyldiimidazole.

EXAMPLE V Conjugation of Radiolabeled Chelate and Chelating Compound andRadiolabeling with Isotopes

Radiolabeling of chelating compounds with (a) ^(99m) Tc and (b)^(186/188) Re and conjugation with antibody (and fragments).

(a) To 100 μL of solution containing 5 mg of sodium gluconate and 0.1 mgof SnCl₂ in water, 500 μL of ^(99m) TcO₄ ⁻ (pertechnetate) is added.After incubation at room temperature for 10 minutes to form Tc-gluconatecomplex, 100 μg of a chelating compound (dissolved in i-propanol:aceticacid 90:10, a 1 mg/mL solution), 80 μL of 0.2N HCl, and 200 μL ofi-propanol are added in that order. The chelating compound may be one ofthe four chelating compounds 11, 26, 38, or 46 prepared in EXAMPLES I toIV above. The reaction mixture is heated to 37° C. for 15 minutes, thencooled in ice for 5 minutes. To the above chelate, 100 μL of bicarbonatebuffer is added, so that the pH of the solution is about 6.0. Next, 400μL of an antibody (or fragment (5 mg/mL)) is added in the same buffer.The antibody is a monoclonal antibody (or fragments thereof) designatedas NR-ML-05 (specific for melanoma cells), NR-LU-10 (a pancarcinomamonoclonal antibody), NR-CO-02 (specific for carcinoembryonic antigen(CEA) and colon carcinoma), or NR-CE-01 (anti-CEA). The reactionmixtures are incubated at room temperature for 1/2-1 hour as necessary.ITLC procedure (Nuclear Medicine Technology and Techniques, ed. Bernlet,D., Longan, J., and Wells, L., The C. V. Mosby Co., St. Louis, Mo.,(1981); pp. 172-174) using 12% TCA is used to determine the percentageof chelate attached to the protein.

(b) The ¹⁸⁸ Re chelate of the ligand is prepared in a similar procedure.Sodium perrhenate (3 mL, 15 mCi, produced from a ¹⁸⁸ W/¹⁸⁸ Re generator)is added to a vial containing a lyophilized mixture comprising citricacid, 75 mg; stannous chloride, 0.75 mg; gentisic acid, 0.25 mg andlactose, 100 mg. The vial is agitated gently to mix the contents, thenincubated at room temperature for 10 minutes to form a ¹⁸⁸ Re-citratecomplex. To a separate vial containing the 0.50 mg of the chelatingagent, 0.50 mL of i-propanol is added and the vial is agitated for 2minutes to completely dissolve the compound. Then 0.3 mL of thissolution is transferred to the vial containing the Re-citrate complex.The reaction mixture is heated at 37° C. for 15 minutes, then cooled inice for 5 minutes. The incubation with antibody (or its fragments) iscarried out exactly as described in procedure (a) above usingappropriate volume of bicarbonate buffer.

In both cases, the final purification of the antibody-chelate conjugateis achieved by passing the conjugate through a Sephadex-G₂₅ or aQAE-column. The purity of the conjugate is usually over 97% beforeadministration to test animals and to humans.

Preparation of antibody-chelating compound conjugate followed bylabeling with ^(99m) Tc and ¹⁸⁸ Re:

Preparation of the conjugate: The antibody conjugation reaction iscontained in a final volume of 4 mL: 0.1 mg (62 μmol) of the chelatingcompound, 1.1 mg of the monoclonal antibody (IgG, 7.3×10⁻⁹ moles), 1-2mL of distilled dimethylformamide (if necessary to solubilize thechelating compound), 0.05M of borate or 0.5M bicarbonate buffer at pH8.5. After stirring for 90 minutes at room temperature, 4.4 mL of 5Nsodium chloride is added. After additional 30 minutes, the reactionmixture is centrifuged to remove any particulates and the supernatantfractioned by gel filtration column chromatography. The column eluent ismonitored at 280 nm and the fractions containing monomeric antibodyconjugate are pooled and concentrated in an Amicon stirred cell (30,000molecular weight cutoff).

(i) Technetium-99m labeling of antibody chelating compound conjugatewith ^(99m) Tc-tartrate.

Stannous tartrate kits are prepared from degassed solutions of 0.5 mLdisodium tartrate (150 mg/mL) and 0.1 mL stannous chloride (1.0 mg/mL inethanol) in an evacuated vial under nitrogen atmosphere. To a stannoustartrate kit, sodium pertechnetate 0.5 mL (about 15 mCi) is added andheated at 50° C. for 10-15 minutes. After cooling to room temperature,quality control for ^(99m) Tc tartrate and insoluble ^(99m) Tc iscarried out on Gelman ITLC using methyl ethyl ketone and 0.01M sodiumtartrate pH 7.0 eluants, respectively. ^(99m) Tc tartrate formation istypically 98-99% with soluble ^(99m) Tc values ranging from 0.1 to 0.2%.

In an evacuated vial, 100 μL saline, 200 μL of sodium phosphate (0.2M,pH 8.0) and 200 μL of antibody-chelating compound conjugate (1.9 mg/mL)are added successively. Immediately after adding the conjugate, 250 μLof ^(99m) Tc tartrate (about 3 to 5 mCi) is added and heated at 37° C.for 1 hour. Percent technetium bound to protein and the formation ofpertechnetate are determined by ITLC using 50% MeOH:10% ammonium acetate(1:1) and 1-butanol eluents, respectively.

(ii) Rhenium-188 labeling of antibody-chelating compound conjugate withRe-citrate.

The ¹⁸⁸ Re chelate is prepared in a similar procedure. Sodium perrhenate(3 mL, 15 mCi, produced from a ¹⁸⁸ W/¹⁸⁸ Re generator) is added to avial containing a lyophilized mixture comprising citric acid, 75 mg;stannous chloride, 0.75 mg; gentisic acid, 0.25 mg and lactose, 100 mg.The vial is agitated gently to mix the contents, then incubated at roomtemperature for 10 minutes to form a ¹⁸⁸ Re-citrate complex. Thereaction mixture is heated at 75° C. for 15 minutes, then cooled in leefor 5 minutes to prepare the Re-citrate complex for labeling of theconjugate.

Labeling of the conjugate is carried out in a similar proceduredescribed for Tc in (i).

EXAMPLE VI Diagnostic and Therapeutic Kits

(A) Diagnostic Kit.

(i) Pre-Formed Approach

A diagnostic kit containing reagents for preparation of a ^(99m)Tc-radiolabeled protein conjugate is used as follows. The procedures areconducted under conditions which ensure the sterility of the product(e.g., sterile vials and sterilized reagents are used where possible,and reagents are transferred using sterile syringes). Proper shieldingwas used once the radioisotope is introduced.

One mL of sterile water for injection is added to a sterile vialcontaining a stannous gluconate complex (50 mg sodium gluconate and 1.2mg stannous chloride dihydrate, available from Merck Frosst, Canada, indry solid form) and the vial is gently agitated until the contents aredissolved. A sterile insulin syringe is used to inject 0.1 mL of theresulting stannous gluconate solution into an empty sterile vial. Sodiumpertechnetate (0.75 mL, 75-100 mCi, from a ⁹⁹ Mo/⁹⁹ Tc generatoravailable from DuPont, Mediphysics, Mallinckrodt, or E. R. Squibb) isadded, and the vial is agitated gently to mix the contents, thenincubated at room temperature for 10 minutes to form a ^(99m)Tc-gluconate complex. In an alternative procedure for providing the^(99m) Tc-gluconate exchange complex, the kit includes a vial containinga lyophilized preparation comprising 5 mg sodium gluconate, 0.12 mgstannous chloride dihydrate, about 0.1 mg gentisic acid as a stabilizercompound, and about 20 mg lactose as a filler compound. The amount ofgentisic acid may vary, with the stabilizing effect generally increasingup to about 0.1 mg. Interference with the desired reactions may occurwhen about 0.2 mg or more gentisic acid is added. The amount of lactosealso may vary, with amounts between 20 and 100 mg, for example, beingeffective in aiding lyophilization. Addition of stabilizer and a fillercompound is especially important when the vial contained theserelatively small amounts of sodium gluconate and stannous chloride(compared to the alternative embodiment above). One mL of sodiumpertechnetate (about 100 mCi) is added directly to the lyophilizedpreparation. The vial is agitated gently to mix the contents, thenincubated as described above to form the ^(99m) Tc-gluconate complex.

A separate vial containing 0.3 mg of a chelating compound of the presentinvention in dry solid form is prepared by dispensing a solution of 0.3mg chelating agent in i-propanol into the vial, then removing thesolvent under N₂ gas, and the resulting vial containing the chelatingcompound is provided in the kit. To this vial is then added 0.87 mL of100% i-propanol, and the vial is gently shaken for about 2 minutes tocompletely dissolve the chelating agent. Next, 0.58 mL of this solutionof the chelating agent is transferred to a vial containing 0.16 mL ofglacial acetic acid/0.2N HCl (2.14), and the vial is gently agitated. Ofthis acidified solution, 0.5 mL is transferred to the vial containingthe ^(99m) Tc-gluconate complex, described above. After gentle agitationto mix, the vial is incubated in a 37° C. water bath for 15 minutes,then immediately transferred to a 0° C. ice bath for 2 minutes.

To a separate vial containing 10 mg of the Fab fragment of a monoclonalantibody in 0.5 mL of phosphate-buffered saline, is added 0.37 mL of1.0M sodium bicarbonate buffer, pH 10.0. The Fab fragment is generatedby treating the monoclonal antibody with papain according toconventional techniques. The vial is then gently agitated.

The vial containing the acidified solution of the ^(99m) Tc-labeledchelate (see above) is removed from the ice bath, 0.1 mL of the sodiumbicarbonate buffer is added, and the vial is agitated to mix.Immediately, the buffered antibody solution (above) is added, gentlyagitated to mix and incubated at room temperature for 20 minutes toallow conjugation of the radiolabeled chelate to the antibody.

A column containing an union exchanger, either DEAE-Sephadex orQAE-Sephadex, is used to purify the conjugate. The column is preparedunder aseptic conditions as follows. Five 1 mL QAE-Sephadex columns areconnected end-to-end to form a single column. Alternatively, a single 5mL QAE-Sephadex column may be used. The column is washed with 5 mL of 37mM sodium phosphate buffer, pH 6.8. A 1.2μ filter (available fromMillipore) is attached to the column, and a 0.2μ filter is attached tothe 1.2μ filter. A 22-gauge sterile, nonpyrogenic needle was attached tothe 0.2μ filter.

The reaction mixture is drawn up into a 3 mL or 5 mL syringe, and anyair bubbles are removed from the solution. After removal of the needle,the syringe is connected to the QAE-Sephadex column on the end oppositethe filters. The needle cap is removed from the 22-gauge needle attachedto the filter end of the column and the needle tip is inserted into asterile, nonpyrogenic test tube. Slowly, over 2 minutes, the reactionmixture is injected into the column. The eluant collected in the testtube is discarded. The now empty syringe on top of the column isreplaced with a 5 mL syringe containing 5 mL of 75 mM (0.45%) sodiumchloride solution (from which air bubbles had been removed). The needleat the other end of the column is inserted aseptically into a sterile,nonpyrogenic 10 mL serum vial. Slowly, over 2 minutes, the NaCl solutionis injected into the column, and the eluent is collected in the serumvial.

The total radioactivity in the serum vial is measured using a dosecalibrator. The yield of the radiolabeled antibody is normally in the60% range. The contents of the serum vial are drawn up into a sterile,pyrogen-free, 30 cc syringe and diluted to a total volume of 30 mL withsterile 0.9% NaCl for injection into a human patient. A quality controltest is normally performed on a 0.01 mL aliquot before injection byinstant thin layer chromatography.

If the radiochemical purity is less than 85%, the material should not beinjected into a human patient. Using this procedure, radiochemicalpurities generally range from about 90% to 99%. The total amount ofradioactivity also is measured prior to injection. In general, from 10to 30 mCi will be administered to a human patient.

Prior to administering the radiolabeled Fab fragment (the diagnosticradiolabeled antibody fragment), an irrelevant antibody and an unlabeledspecific antibody may be administered to the patient to improve thediagnostic images, as described above. The irrelevant antibody and theunlabeled specific antibody, are provided in the kit in separate vials.

The entire 30 mL sample containing the radiolabeled antibody fragment isadministered to a patient by intravenous infusion. The infusion iscompleted in from about 5 minutes to about 15 minutes. The antibodyfragment concentration in the sample is 0.33 mg/mL.

(ii) Post-Formed Approach

Stannous tartrate kits are prepared from degassed solutions of 0.5 mLdisodium tartrate (150 mg/mL) and 0.1 mL stannous chloride (1.0 mg/mL inethanol) in an evacuated vial under nitrogen atmosphere. To a stannoustartrate kit, sodium pertechnetate 0.5 mL (about 15 mCi) is added andheated at 50° C. for 10-15 minutes. After cooling to room temperature,quality control for ^(99m) Tc-tartrate and insoluble ^(99m) Tc iscarried out on Gelman ITLC using methyl ethyl ketone and 0.01M sodiumtartrate pH 7.0 eluents, respectively. ^(99m) Tc-tartrate formation istypically 98-99% with soluble ^(99m) Tc values ranging from 0.1 to 0.2%.

In an evacuated vial, 100 μL saline, 200 μL of sodium phosphate (0.2M,pH 8.0) and 200 μL of antibody-chelating compound conjugate (1.9 mg/mL)are added successively. Immediately after adding the conjugate, 250 μLof ^(99m) Tc-tartrate (about 3 to 5 mCi is added and heated at 37° C.for 1 hour. Percent technetium bound to protein and the formation ofpertechnetate are determined by ITLC using 50% MeOH:10% ammonium acetate(1:1) and 1-butanol eluants, respectively.

(B) Therapeutic Kit

(i) Pre-Formed Approach

The ¹⁸⁸ Re chelate is prepared in a similar procedure. Sodium perrhenate(3 mL, 15 mCi, produced from a ¹⁸⁸ W/¹⁸⁸ Re generator) is added to avial containing a lyophilized mixture comprising citric acid, 75 mg;stannous chloride, 0.75 mg; gentisic acid, 0.25 mg and lactose, 100 mg.The vial is agitated gently to mix the contents, then incubated at 75°C. for 15 minutes and cooled to room temperature to form ¹⁸⁸ Re-citratecomplex. To a separate vial containing the 0.50 mg of the chelatingagent, 0.50 mL of i-propanol is added and the vial is agitated for 2minutes to completely dissolve the compound. Then 0.3 mL of thissolution is transferred to the vial containing the Re-citrate complex,and incubated at room temperature for about 1 hour to produce thedesired ¹⁸⁸ Re-chelate.

A column containing a C₁₈ reversed phase low-pressure material (BakerC₁₈ cartridges) is used to purify the ¹⁸⁸ Re-labeled chelate. Afterconditioning of the cartridge with ethanol and water, the sample isloaded and washed with three times 2 mL of water and three times 2 mL of20% ethanol/0.01M phosphate buffer. The column is then dried in vacuoand eluted with two times 1.0 mL acetonitrile.

The chelate is then conjugated to a Fab fragment of a monoclonalantibody. A buffered solution of the antibody fragment (5 mg/mL, 0.5 mL)is added to the purified ¹⁸⁸ Re-labeled chelate, followed by 0.5 mL of0.5M carbonate/bicarbonate buffer pH 9.50. The reaction is kept at roomtemperature for 15 minutes, then 25 mg of L-lysine, 0.1 mL, is added andthe reaction is pursued at room temperature for 15 minutes more.

A column containing Sephadex-G₂₅ material is used to purify the ¹⁸⁸Re-chelate-antibody conjugate. The reaction mixture is loaded on top ofthe column, and 1.2 mL aliquots are collected using PBS buffer to rinsethe reaction vial and elute the ¹⁸⁸ Re conjugate in the third and fourthfractions. The purity of the ¹⁸⁸ Re conjugate is usually greater than97%. The conjugate is then further diluted with PBS, and radioactivityis measured prior to injection into the test animals and human subjects.

(ii) Post-Formed Approach

Sodium perrhenate (3 mL, 15 mCi, produced from a ¹⁸⁸ W/¹⁸⁸ Re generator)is added to a vial containing a lyophilized mixture comprising citricacid, 75 mg; stannous chloride, 0.75 mg; gentisic acid, 0.25 mg andlactose, 100 mg. The vial is agitated gently to mix the contents, thenincubated at room temperature for 10 minutes to form a ¹⁸⁸ Re-citratecomplex. The reaction mixture is heated at 75° C. for 15 minutes, thencooled in ice for 5 minutes to prepare the Re-citrate complex ready forlabeling of the conjugate. Labeling of the ligand-antibody conjugate iscarried out in a procedure similar to procedure A(ii).

EXAMPLE VII Imaging of Tumors in Humans

Antibody fragments radiolabeled with ^(99m) Tc according to the methodof the invention are injected into human patients to detect tumor sites(melanoma, lung, colon, etc. depending on the antibody used) within thebody. The antibody fragments used are F(ab)')₂, Fab' or Fab fragments ofa monoclonal antibody specific for an antigen of the particular targettumor. The fragments were generated by standard techniques (i.e., pepsintreatment of the monoclonal antibody to generate the F(ab')₂ fragment,papain treatment of the monoclonal antibody to generate the Fabfragment, and treatment with a reducing agent such as dithiothreitol togenerate the Fab' fragment).

Each patient receives a ^(99m) Tc-chelate-antibody fragment conjugateprepared by the procedures described in EXAMPLE V above. Theradiolabeled antibody fragments are purified, and a quality control testis performed, as described in EXAMPLE VI(A)(i). Approximately 40 minutesto 1 hour and 30 minutes prior to infusion of the radiolabeled antibody,each patient receives 41 to 50 mg of an irrelevant antibody in 12 to 20mL of sterile saline by intravenous infusion. In addition, each patientreceives 7.5 mg of a nonradiolabeled specific antibody in 20 mL ofsterile saline by intravenous infusion either simultaneously with, orapproximately 5 minutes prior to infusion of the radiolabeled specificantibody. The nonradiolabeled specific antibody is exactly the same asthe one used for radiolabeling, generally in the form of a wholeantibody or a F(ab')₂ fragment thereof. The irrelevant antibody is amonoclonal antibody designated NR2AD, which is a murine IgG_(2a)immunoglobulin that is designed as an anti-idiotype that binds to asingle patient's B-cell lymphoma and to no other human tissue.

Into each patient is injected 20 to 30 mL of sterile saline comprisingthe radiolabeled antibody fragment, by intravenous infusion. The patientreceives from 11.4 mCi to about 30 mCi of ^(99m) Tc radioisotope. Thedesired upper limit of radioisotope administered is 30 mCi, and theminimum for effective imaging of tumors is generally about 10 mCi. Thetotal amount of protein in the administered solutions ranges from 2.5mgs to 10 mgs. Imaging by gamma camera is performed at varioustimepoints, including a baseline image: immediately following infusionof the radiolabeled antibody and at timepoints less than 20 hourspost-infusion such that "background" has substantially cleared but theradionuclide is still at an imageable level. The percentage of the totalinjected dose of radioactivity (in cpm) which had localized in each ofthe various tissue types sampled are calculated. The ratio of theradioactivity found in tumor sites to the radioactivity found in theother types of tissue are also calculated. The value for "percentinjected dose per mg" for the tumor tissue in a particular patient isdivided by the value for "percent injected dose per mg" for eachnontumor tissue sample extracted from the patient to give thetumor/tissue ratio for each nontumor tissue sample.

EXAMPLE VIII Biodistribution Studies

Biodistribution Studies in Mice for ^(99m) Tc-labeled and ¹⁸⁸ Re-labeledMonoclonal Antibody Fragments

Antibody fragments radiolabeled with ^(99m) Tc or ¹⁸⁸ Re are injectedinto tumor-bearing mice, and biodistribution of the radionuclidemetal-chelate-protein conjugate is analyzed 20 hours after injectionaccording to the method of Hwang, et al., Cancer Res., 45:4150-4155(1985). The antibody fragment is a Fab fragment of one of theabove-described monoclonal antibodies (NM-ML-05, NR-LU-10, NR-CO-02, orNR-CE-01). The data is collected in terms of the percentage of theinjected radioactivity per gram of each specified tissue type andtumor/tissue ratio of injected radioactivity. The tissue types are asfollows: tail; tumor; skin; muscle; bone; lung; liver; spleen; stomach;thyroid; kidney; and intestine. A high percentage of the injectedradioactivity is localized at the tumor site in each mouse.

While the invention has been described in conjunction with preferredembodiments, one of ordinary skill after reading the foregoingspecification will be able to effect various changes, substitutions ofequivalents, and alterations to the subject matter set forth herein.Hence, the invention can be practiced in ways other than thosespecifically described herein. It is therefore intended that theprotection granted by Letters Patent hereon be limited only by theappended claims and equivalents thereof.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A conjugate comprising aprotein, protein fragment or polypeptide bound to a chelating compoundcomprising a first site at which a complex of a radionuclide metalforms, a second site at which a chelate of the radionuclide metal forms,and a conjugation group adapted to bind the chelating compound to theprotein, wherein said first site comprises two or more atoms selectedfrom oxygen, nitrogen and phosphorous, said second site comprises aheteroatom chain containing at least one sulfur atom and at least twonitrogen atoms, and the complex formed at said first site has a fasterrate of formation and a lower thermodynamic stability than said chelateformed at said second site, such that when a radionuclide metal iscombined with said compound, the complex at the first site formsinitially, and the radionuclide metal subsequently is transferred to thesecond site to form the chelate.
 2. The conjugate of claim 1 whereinsaid protein is a monoclonal antibody or an antigen binding fragmentthereof.
 3. The conjugate of claim 2 wherein said monoclonal antibodybinds cancer cells.
 4. The conjugate of claim 1 wherein the radionuclidemetal is transferred to the second site by incubating the compound afterthe complex has formed.
 5. The conjugate of claim 4 wherein the compoundis heated to a temperature between ambient temperature and about 37° C.6. The conjugate of claim 1 wherein the first site comprises two or moreatoms chosen from oxygen, nitrogen, and phosphorous in the form of anoxide, wherein said atoms interact with the radionuclide metal to formthe complex.
 7. The conjugate of claim 1 wherein the second sitecomprises a heteroatom chain containing at least four donor atoms chosenfrom sulfur, nitrogen, and oxygen, wherein coordinate covalent bondsform between each of said donor atoms and the radionuclide metal to formthe chelate.
 8. The conjugate of claim 7 wherein said donor atomsinclude at least one divalent sulfur atom and at least two nitrogenatoms, the sulfur atom being positioned at one terminus of theheteroatom chain, and from six to seven carbon atoms positioned so thatat least two carbon atoms are positioned between any two of the donoratoms.
 9. The conjugate of claim 8 wherein said donor atoms are twonitrogen atoms and two sulfur atoms.
 10. The conjugate of claim 8wherein said donor atoms are three nitrogen atoms and one sulfur atom.11. The conjugate of claim 8 wherein at least one sulfur donor atom,together with a protective group attached thereto, defines ahemithioacetal group.
 12. The conjugate of claim 1 wherein said firstsite is bound to said second site through a flexible divalent linker.13. The conjugate of claim 12 wherein said linker comprises from two tosix methylene groups in a chain.
 14. The conjugate of claim 1 whichfurther comprises a conjugation group which binds the protein to thechelating compound, wherein the conjugation group is attached through aspacer to a carbon or a nitrogen atom of the chelating compound.
 15. Theconjugate of claim 1 represented by the structural formula: ##STR16## 1)wherein: a) T is a sulfur-protecting group;b) R₀ is S--T, O⁻, ═O or##STR17## c) the R groups designated R₁ through R₁₁ are independentlyselected from COOH, R₁₄ and R₁₅ --Z--Pr, wherein any two of said Rgroups, when bonded to the same carbon atom, may be taken together toform an oxo group, with the provisos that:i) R₇ and R₈ taken together,and R₉ and R₁₀ taken together, are not both simultaneously oxo; ii) R₂and R₃ taken together, and R₄ and R₅ taken together, are not bothsimultaneously oxo; iii) R₄, R₅, R₉, and R₁₀ may all be taken togetherto form a hydrocarbon ring; iv) R₆ is hydrogen when either R₂ and R₃ orR₄ and R₅ represent an oxo group; v) R₁₁ is hydrogen when either R₇ andR₈ or R₉ and R₁₀ represent an oxo group; vi) each of R₇, R₈, R₉, and R₁₀are hydrogen when R₁₁ is R₁₅ --Z--Pr or R₁₆ ; vii) each of R₂, R₃, R₄,and R₅ are hydrogen when R₆ is R₁₅ --Z--Pr or R₁₆ ; viii) R₆ and R₁₁cannot be COOH; d) R₁₂ and R₁₃ are (1) independently R₁₄ or R₁₅ --Z--Prwhen R₀ is either S--T or ##STR18## or (2) taken together to form oxowhen R₀ is either O⁻, ═O or ##STR19## with the proviso that when R₁₂ andR₁₃ are oxo and R₀ is ##STR20## and one of these two R₁ groups is R₁₅--Z--Pr or R₁₆, the other R₁ group is hydrogen; e) R₁₄ is hydrogen,lower alkyl, or R₁₆ ; f) n is 0 or 1 provided that n equals 1 not morethan once; g) R₁₅ is a divalent spacer selected from substituted orunsubstituted lower alkyl, which may additionally comprise one or moregroups selected from --O--, --NH--, --NR--, --CO--, --CO₂ --, --CONH--,--S--, --SO--, --SO₂ --, --CO₂ NH--, and --SO₂ NR--, where R is selectedfrom C₁ -C₃ alkyl; h) Z is a functional group suitable for reacting witha protein, protein fragment, or polypeptide under conditions thatpreserve biological activity of the protein, protein fragment, orpolypeptide; i) Pr is a protein, protein fragment or polypeptide; 2)wherein said compound comprises at least one substituent R₁₅ --Z--Pr;and 3) wherein at least one of the groups R₁ -R₁₄ is substituent R₁₆having the formula: ##STR21## wherein: a) one or more R₁₇ groups arebonded to any carbon or nitrogen atom and are selected from the grouphydrogen, lower alkyl, or R₁₅ --Z--Pr;b) X is a radical selected fromthe group consisting of fully substituted carbon, nitrogen, oxygen, sp²or sp carbon, or amide or imide nitrogen; c) p in an integer, 2, 3, 4,5, or 6 provided that:i) when p is 2 or 3, X is fully substitutedcarbon; ii) when p is 4, X is fully substituted carbon, nitrogen, oroxygen, further provided that at least three radicals X are fullysubstituted carbon; iii) when p is 5 or 6, at least three radicals X arefully substituted carbon and further provided that only one radical Xmay be sp² or sp carbon, amide or imide nitrogen; and d) Q representsthe first site.
 16. The conjugate of claim 15 wherein Q is selected fromthe group consisting of iminodiacetate, alkyl phosphonate, alkyldiphosphonate, N-glycine, aminoalkylpolyacetate,alkylhydroxycarboxylate, polyhydroxyaminoalkanes,alkylaminohydroxycarboxylate, and alkyldihydroxydicarboxylate.
 17. Theconjugate of claim 15 wherein Q is selected from the group consisting ofpolyhydroxycarboxylates, gluconate, tartrate, alkyl phosphonate, alkyl,disphosphonate, gluconamide, N-glycine, N-3-aminopropanoate, α-hydroxyacids, α-hydroxy-β-amino acids, α-hydroxy-α-amino acids, deoxyaminouronic acids, β-diketones or enol equivalents thereof, ##STR22## andderivatives thereof; wherein R is C₁ -C₅ lower alkyl and R₁₇ ishydrogen, lower alkyl or R₁₅ --Z--Pr, wherein R₁₅ is a divalent spacerand Z is a group reactive with a protein.
 18. The conjugate of claim 15wherein Z is selected from the group consisting of active esters,isothiocyanate groups, maleimide groups, amine groups, and halomethylketones.
 19. The conjugate of claim 15 wherein one or more substituentsT is a protecting group which, when taken together with the sulfur atomto which it is attached, defines a hemithioacetal.
 20. The conjugate ofclaim 19 wherein said hemithioacetal is selected from the groupconsisting of ethoxyethyl, tetrahydropyranyl, 2-methyltetrahydropyranyl, 6-carboxy tetrahydropyranyl, tetrahydrofuranyl, and2-methyl tetrahydrofuranyl.
 21. The conjugate of claim 15 wherein thecompound is represented by one of the structural Formulae II-XIV:##STR23## wherein: a) when R₆ is R₁₆ or R₁₅ --Z--Pr, each R₁ groupattached to a carbon atom adjacent to --N--R₆ is hydrogen; and,b) whenR₁₁ is R₁₆ or R₁₅ --Z--Pr, each R₁ group attached to a carbon atomadjacent to --N--R₁₁ is hydrogen.
 22. The conjugate of claim 15 whereinthe compound has the following structural formula: ##STR24## wherein Zrepresents an active ester group or an isothiocyanate group.
 23. Aconjugate of claim 1 which further comprises a radionuclide metalchelated with the first or second site of the chelating compound. 24.The conjugate of claim 23 wherein the radionuclide metal is selectedfrom the group consisting of ^(99m) Tc, ¹⁸⁸ Re, ¹⁸⁶ Re, ⁶⁷ Cu, ⁶⁴ Cu,²¹² Pb, ²¹² Bi, ¹⁰⁵ Rd, ⁹⁷ Ru, and ¹⁰⁹ Pd.
 25. The conjugate of claim 24wherein the radionuclide metal is selected from ^(99m) Tc, ¹⁸⁸ Re, and¹⁸⁶ Re.